Process for producing isoprenoid compounds by microorganisms and a method for screening compounds with antibiotic or weeding activity

ABSTRACT

The present invention provides a process for producing isoprenoid compounds or proteins encoded by DNA using DNA that contains one or more of the DNA encoding proteins having activity to improve efficiency in the biosynthesis of isoprenoid compounds effective in pharmaceuticals for cardiac diseases, osteoporosis, homeostasis, prevention of cancer, and immunopotentiation, health food and anti-fouling paint products against barnacles; the DNA; the protein; and a method for screening a substance with antibiotic and weeding activities comprising screening a substance inhibiting enzymatic reaction on the non-mevalonate pathway.

This application is a divisional of application Ser. No. 10/938,613filed Sep. 13, 2004, which in turn is a divisional of application Ser.No. 09/673,198 filed Oct. 12, 2000 (now U.S. Pat. No. 6,806,076), whichin turn is a National Phase of PCT Application No. PCT/JP99/01987 filedApr. 14, 1999.

TECHNICAL FIELD

The present invention relates to a method for producing isoprenoidcompounds using a transformant derived from a prokaryote; and a methodfor screening substances having antibiotic or weeding activity involvedin a non-mevalonate pathway.

BACKGROUND ART

Isoprenoid is a general term for compounds having isoprene unitconsisting of 5 carbon atoms as a backbone structure. Isoprenoid isbiosynthesized by polymerization of isopentenyl pyrophosphate (IPP).Various kinds of isoprenoid compounds are present in nature and many ofthem are useful for humans.

For example, ubiquinone plays an important role in vivo as an essentialcomponent of the electron transport system. The demand for ubiquinone isincreasing not only as a pharmaceutical effective against cardiacdiseases, but also as a health food in Western countries.

Vitamin K, an important vitamin involved in the blood coagulationsystem, is utilized as a hemostatic agent. Recently it has beensuggested that vitamin K is involved in osteo-metabolism, and isexpected to be applied to the treatment of osteoporosis. Phylloquinoneand menaquinone have been approved as pharmaceuticals.

In addition, ubiquinone and vitamin K are effective in inhibitingbarnacles from clinging to objects, and so would make an excellentadditive to paint products to prevent barnacles from clinging.

Further, compounds called carotenoids having an isoprene backboneconsisting of 40 carbon atoms have antioxidant effect. Carotenoids suchas β-carotene, astaxanthin, and cryptoxanthin are expected to possesscancer preventing and immunopotentiating activity.

As described above, isoprenoid compounds include many effectivesubstances. Establishment of an economical process for producing thesesubstances will be a huge benefit to the medical world and society.

The process for producing isoprenoid compounds through fermentation hasalready been examined, and examination of culture conditions, strainbreeding by mutagenesis, and improvement of yield by genetic engineeringtechniques have been tested. However, the practical results are limitedto individual types of compounds, and there is no known method effectivefor the isoprenoid compounds in general.

Isopentenyl pyrophosphate (IPP), a backbone unit of isoprenoidcompounds, has been proved to be biosynthesized from acetyl-CoA viamevalonic acid (mevalonate pathway) in eukaryotes, such as an animal andyeast.

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase is considered to be arate-limiting enzyme in the mevalonate pathway [Mol. Biol. Cell, 5, 655(1994)]. A test in yeast to improve the yield of carotenoids byoverexpression of HMG-CoA reductase has been conducted [Misawa, et al.,Summaries of Lectures on Carotenoids, 1997].

There is no knowledge which proves the presence of the mevalonatepathway in prokaryotes. In many prokaryotes, another pathway, thenon-mevalonate pathway, has been found in which IPP is biosynthesizedvia 1-deoxy-D-xylulose 5-phosphate produced by condensation of pyruvicacid and glyceraldehyde 3-phosphate [Biochem. J., 295, 517 (1993)]. Itis suggested that 1-deoxy-D-xylulose 5-phosphate is converted to IPP via2-C-methyl-D-erythritol 4-phosphate in an experiment using ¹³C-labelledsubstrate [Tetrahedron Lett. 38, 4769 (1997)].

In Escherichia coli, a gene encoding an enzyme, 1-deoxy-D-xylulose5-phosphate synthase (DXS) which allows biosynthesis of1-deoxy-D-xylulose 5-phosphate by condensation of pyruvic acid andglyceraldehyde 3-phosphate, is identified [Proc. Natl. Acad. Sci. USA,94, 12857 (1997)]. Said gene is contained in an operon consisting offour ORFs that include ispA encoding farnesyl pyrophosphate synthase.

Further in Escherichia coli, the presence of the activity to convert1-deoxy-D-xylulose 5-phosphate to 2-C-methyl-D-erythritol 4-phosphate isknown [Tetrahedron Lett. 39, 4509 (1998)].

At present there are no known description nor suggestion to improveyield of an isoprenoid compound by genetically engineering these genescontained in the operon.

Although knowledge about the non-mevalonate pathway in prokaryotes hasgradually increased, most enzymes involved therein and genes encodingthese enzymes still remain unknown.

In photosynthetic bacteria, there is a known process for effectivelyproducing ubiquinone-10 by introducing a gene for an enzyme ubiC (uviCgene), which converts chorismate into 4-hydroxybenzoate, and a gene forp-hydroxybenzoate transferase (ubiA) (Japanese Unexamined PatentApplication 107789/96). However, there is no example which improved theproductivity of isoprenoid compounds by genetically engineering genesfor enzymes involved in the non-mevalonate pathway.

Moreover, there is no knowledge about how prokaryotes will be influencedwhen the reaction on the non-mevalonate pathway is inhibited bymutagenesis or treating with drugs.

DISCLOSURE OF THE INVENTION

The object of this invention is to provide a process for producingisoprenoid compounds comprising integrating DNA into a vector whereinthe DNA contains one or more DNA involved in biosynthesis of isoprenoidcompounds useful in pharmaceuticals for cardiac diseases, osteoporosis,homeostasis, prevention of cancer, and immunopotentiation, health foodand anti-fouling paint products against barnacles, introducing theresultant recombinant DNA into a host cell derived from prokaryotes,culturing the obtained transformant in a medium, allowing thetransformant to produce and accumulate isoprenoid compounds in theculture, and recovering the isoprenoid compounds from said culture; aprocess for producing proteins comprising integrating DNA into a vectorwherein the DNA contains one or more DNA encoding a protein havingactivity to improve efficiency in the biosynthesis of isoprenoidcompounds, introducing the resultant recombinant DNA into a host cell,culturing the obtained transformant in a medium, allowing thetransformant to produce and accumulate said protein in the culture, andrecovering said protein from the culture; the protein; and DNA encodingthe protein. A further object of this invention is to provide a methodof screening a substance having antibiotic and/or weeding activities,which comprises screening the substance inhibiting enzymatic reaction onthe non-mevalonic acid pathway.

The inventors have completed the invention by finding that theproductivity of isoprenoid can be improved by screening DNA capable ofimproving the productivity for isoprenoid in prokaryotes, andintroducing the obtained DNA into prokaryotes.

That is, the first invention of the present application is a process forproducing isoprenoid compounds comprising integrating DNA into a vectorwherein the DNA contains one or more DNA selected from the following(a), (b), (c), (d), (e) and (f):

-   (a) a DNA encoding a protein having activity to catalyze a reaction    to produce 1-deoxy-D-xylulose 5-phosphate from pyruvic acid and    glyceraldehyde 3-phosphate,-   (b) a DNA encoding farnesyl pyrophosphate synthase,-   (c) a DNA encoding a protein that has an amino acid sequence of SEQ    ID NO:3, or a protein that has an amino acid sequence wherein one to    several amino acid residues are deleted, substituted or added in the    amino acid sequence of SEQ ID NO:3 and has activity to improve    efficiency in the biosynthesis of isoprenoid compounds,-   (d) a DNA encoding a protein that has an amino acid sequence of SEQ    ID NO:4, or a protein that has an amino acid sequence wherein one to    several amino acid residues are deleted, substituted or added in the    amino acid sequence of SEQ ID NO:4 and has activity to improve    efficiency in the biosynthesis of isoprenoid compounds,-   (e) a DNA encoding a protein having activity to catalyze a reaction    to produce 2-C-methyl-D-erythritol 4-phosphate from    1-deoxy-D-xylulose 5-phosphate, and-   (f) a DNA encoding a protein that can hybridize under stringent    conditions with DNA selected from (a), (b), (c), (d) and (e), and    has activity substantially identical with that of the protein    encoded by the selected DNA;    introducing the resultant recombinant DNA into a host cell derived    from prokaryotes, culturing the obtained transformant in a medium;    allowing the transformant to produce and accumulate isoprenoid    compounds in the culture; and recovering the isoprenoid compounds    from the culture.

Deletions, substitutions or additions of amino acid residues in thisspecification can be carried out by site-directed mutagenesis, which isa technique well-known prior to the filing of this application. Further,the phrase “one to several amino acid residues” means the number ofamino acid residues, which can be deleted, substituted, or added bysite-directed mutagenesis, for example, 1 to 5 amino acid residues.

The protein consisting of an amino acid sequence, which has deletion,substitution or addition of one to several amino acid residues, can beprepared according to the methods described in Molecular Cloning: ALaboratory Manual, Second Edition, ed. Sambrook, Fritsch, and Maniatis,Cold Spring Harbor Laboratory Press, 1989 (hereinafter referred to asMolecular Cloning, Second Edition), Current Protocols in MolecularBiology, John Wiley & Sons (1987-1997), Nucleic Acids Research, 10, 6487(1982), Proc. Natl. Acad. Sci., USA, 79, 6409 (1982), Gene, 34, 315(1985), Nucleic Acids Research, 13, 4431 (1985), and Proc. Natl. Acad.Sci USA, 82, 488 (1985), etc.

The above-mentioned DNA encoding a protein, which catalyzes a reactionto produce 1-deoxy-D-xylulose 5-phosphate from pyruvic acid andglyceraldehyde 3-phosphate, is for example, a DNA encoding a protein,which has an amino acid sequence of SEQ ID NO:1, 26 or 28, or a DNAencoding a protein which has an amino acid sequence wherein one toseveral amino acid residues are deleted, substituted or added in theamino acid sequence of SEQ ID NO:1, 26, or 28 and has activity tocatalyze a reaction to produce 1-deoxy-D-xylulose 5-phosphate frompyruvic acid and glyceraldehyde 3-phosphate.

Examples of such a DNA include a DNA having an nucleotide sequence ofSEQ ID NO:6 or a DNA having a nucleotide sequence of SEQ ID NO:27 or 29.

Examples of a DNA encoding farnesyl pyrophosphate synthase include a DNAencoding a protein having an amino acid sequence of SEQ ID NO:2 or a DNAencoding a protein, which has an amino acid sequence wherein one toseveral amino acid residues are deleted, substituted or added in theamino acid sequence of SEQ ID NO:2 and has enzymatic activity to producefarnesyl pyrophosphate. A specific example is a DNA having a nucleotidesequence of SEQ ID NO:7.

A specific example of the DNA encoding a protein having an amino acidsequence of SEQ ID NO:3 is a DNA having a nucleotide sequence of SEQ IDNO:8.

Further a specific example of the DNA encoding a protein having an aminoacid sequence of SEQ ID NO:4 is a DNA having a nucleotide sequence ofSEQ ID NO:9.

Examples of the DNA encoding a protein having activity to catalyze areaction to produce 2-C-methyl-D-erythritol 4-phosphate from1-deoxy-D-xylulose 5-phosphate include a DNA encoding a protein, whichhas an amino acid sequence of SEQ ID NO:5 or 30, or a DNA encoding aprotein, which has an amino acid sequence wherein one to several aminoacid residues are deleted, substituted or added in the amino acidsequence of SEQ ID NO:5 or 30 and has activity to catalyze the reactionto produce 2-C-methyl-D-erythritol 4-phosphate from -deoxy-D-xylulose5-phoshphate.

Specifically, such a DNA is one having a nucleotide sequence of SEQ IDNO:10 or 31.

The above phrase “DNA . . . that can hybridize under stringentconditions” means a DNA that can be obtained by colony hybridization,plaque hybridization, Southern Blotting or the like using the above DNAor fragments of the DNA as a probe. Such a DNA can be identified byperforming hybridization using a filter with colony- or plaque-derivedDNA, or fragments of the DNA immobilized thereon, in the presence of 0.7to 1.0 mol/l NaCl at 65° C., followed by washing the filter using about0.1 to 2-fold SSC solution (the composition of SSC solution at 1-foldconcentration is consisted of 150 mol/l sodium chloride, 15 mol/l sodiumcitrate) at 65° C.

Hybridization can be carried out according to the methods described inMolecular Cloning, Second Edition. Examples of DNA capable ofhybridizing include a DNA that shares at least 70% or more homology,preferably, 90% or more homology with a nucleotide sequence selectedfrom SEQ ID NOS:1, 2, 3, 4, and 5.

Examples of isoprenoid compounds include ubiquinone, vitamin K₂, andcarotenoids.

The second invention of this application is a protein having activity toimprove efficiency in the biosynthesis of isoprenoid compounds andselected from the following (a), (b) and (c):

-   (a) a protein having an amino acid sequence of SEQ ID NO:3, or a    protein having an amino acid sequence wherein one to several amino    acid residues are deleted, substituted or added in the amino acid    sequence of SEQ ID NO:3-   (b) a protein having an amino acid sequence of SEQ ID NO:4, or a    protein having an amino acid sequence wherein one to several amino    acid residues are deleted, substituted or added in the amino acid    sequence of SEQ ID NO:4, and-   (c) a protein having an amino acid sequence of SEQ ID NO:5, or a    protein having an amino acid sequence wherein one to several amino    acid residues are deleted, substituted or added in the amino acid    sequence of SEQ ID NO:5.

The third invention of this application is a process for producing aprotein having activity to improve efficiency in the biosynthesis ofisoprenoid compounds comprising integrating DNA encoding the proteindescribed in the second invention above into a vector, introducing theresultant recombinant DNA into a host cell, culturing the obtainedtransformant in a medium, allowing the transformant to produce andaccumulate the protein in the culture, and recovering the protein fromthe culture.

The transformants above include microorganisms belonging to the genusEscherichia, Rhodobacter or Erwinia.

The fourth invention of this application is a DNA encoding a proteinhaving activity to improve efficiency in the biosynthesis of isoprenoidcompounds selected from the following (a), (b), (c), (d), (e), (f) and(g):

-   (a) a DNA encoding a protein having an amino acid sequence of SEQ ID    NO:3,-   (b) a DNA encoding a protein having an amino acid sequence of SEQ ID    NO:4,-   (c) a DNA encoding a protein having an amino acid sequence of SEQ ID    NO:5,-   (d) a DNA having a nucleotide sequence of SEQ ID NO:8,-   (e) a DNA having a nucleotide sequence of SEQ ID NO:9,-   (f) a DNA having a nucleotide sequence of SEQ ID NO:10, and-   (g) a DNA that can hybridize with any one of DNA described in (a)    to (f) under stringent conditions.

The fifth invention of this application is a method for screening asubstance having antibiotic activity comprising screening a substancethat inhibits the reaction of a protein having activity of an enzymeselected from those present on the non-mevalonate pathway in which1-deoxy-D-xylulose 5-phosphate biosynthesized from pyruvic acid andglyceraldehyde 3-phosphate is converted to 2-C-methyl-D-erythritol4-phosphate from which isopentenyl pyrophosphate is biosynthesized.

The sixth invention of this application is a method for screening asubstance having weeding activity comprising screening a substance thatinhibits the reaction of a protein having activity of an enzyme selectedfrom those present on the non-mevalonate pathway in which1-deoxy-D-xylulose 5-phosphate biosynthesized from pyruvic acid andglyceraldehyde 3-phosphate is converted to 2-C-methyl-D-erythritol4-phosphate from which isopentenyl pyrophosphate is biosynthesized.

Examples of the proteins in the fifth and sixth inventions above includea protein of the following (a) or (b):

-   (a) a protein having activity to catalyze a reaction to produce    1-deoxy-D-xylulose 5-phosphate from pyruvic acid and glyceraldehyde    3-phosphate, or-   (b) a protein having activity to catalyze a reaction to produce    2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose    5-phosphate.

Examples of the proteins catalyzing the reaction to produce1-deoxy-D-xylulose 5-phosphate from pyruvic acid and glyceraldehyde3-phosphate include a protein having an amino acid sequence of SEQ IDNO:1, or a protein having an amino acid sequence wherein one to severalamino acid residues are deleted, substituted or added in the amino acidsequence of SEQ ID NO:1, and having activity to catalyze1-deoxy-D-xylulose 5-phosphate from pyruvic acid and glyceraldehyde3-phosphate.

Examples of the proteins having activity to catalyze the reaction toproduce 2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose5-phosphate include a protein having an amino acid sequence of SEQ IDNO:5, or a protein having an amino acid sequence wherein one to severalamino acid residues are deleted, substituted or added in the amino acidsequence of SEQ ID NO:5, and having activity to catalyze the reaction toproduce 2-C-methyl-D-erythritol A-phosphate from 1-deoxy-D-xylulose5-phosphate.

The seventh invention of this invention is a substance, which hasantibiotic activity and is obtained by the screening method in the fifthinvention above. Known substances obtained by the above screening methodare not included in this invention.

The inventors have focused on structural similarity of fosmidomycin[3-(N-formyl-N-hydroxyamino)propylphosphonic acid] to2-C-methyl-D-erythritol 4-phosphate, a reaction product from1-deoxy-D-xylulose 5-phosphate reductoisomerase reaction, or a reactionintermediate assumed to be produced in this enzymatic reaction.

Based on the assumption that fosmidomycin has activity to inhibit1-deoxy-D-xylulose 5-phosphate reductoisomerase and antibiotic activity,the inventors have conducted experiments on the screening method of thefifth invention and also described in the following Example 10. As aresult, the inventors found that fosmidomycin is a substance having theactivity to inhibit 1-deoxy-D-xylulose 5-phosphate reductoisomerase andantibiotic activity, and in addition, verified the adequacy of thescreening method of the fifth invention above. However, known compoundfosmidomycin is excluded from this invention.

The eighth invention of this invention is a substance, which has weedingactivity and obtained through the screening method of the sixthinvention above. As described above, any substance that is obtained fromthe screening method and already known is excluded from this invention.

Hereinafter a more detailed explanation of this invention will be given.

I. Cloning of DNA Encoding a Protein Involved in Biosynthesis ofIsoprenoid Compounds

(1) Cloning of DNA Encoding a Protein Involved in Biosynthesis ofIsoprenoid Compounds Using a Nucleotide Sequence of DNA (DXS Gene)Encoding DXS

Using information on previously-determined nucleotide sequences of E.coli chromosome and DXS gene [Proc. Natl. Acad. Sci. USA., 94, 12857(1997)], a DNA region containing DXS gene or genes neighboring DXS geneis obtained by cloning with PCR method from E. coli [Science, 230, 1350(1985)].

An example of information on a nucleotide sequence containing DXS geneis the nucleotide sequence of SEQ ID NO:11.

A concrete example of methods for cloning the DNA region containing DXSgene is as follows.

Escherichia coli, such as an E. coli XL1-Blue strain (available fromTOYOBO CO., LTD.), is cultured in a suitable medium for Escherichiacoli, for example, LB liquid medium [containing 10 g of Bactotrypton(manufactured by Difco Laboratories), 5 g of Yeast extracts(manufactured by Difco Laboratories), 5 g of NaCl per liter of water,and adjusted to pH 7.2] according to standard techniques.

After culturing, cells were recovered from the culture bycentrifugation.

Chromosomal DNA is isolated from the obtained cells according to a knownmethod, described in, for example, Molecular Cloning, Second Edition.

Using information on a nucleotide sequence of SEQ ID NO:11, a senseprimer and an antisense primer, which contain DXS gene or a nucleotidesequence corresponding to the DNA region of genes neighboring DXS gene,are synthesized with a DNA synthesizer.

To introduce the amplified DNA fragments into a plasmid afteramplification with PCR, it is preferable to add recognition sitesappropriate for restriction enzymes, e.g., BamHI, and EcoRI to the 5′ends of sense and antisense primers.

Examples of a combination of the sense and antisense primers include aDNA having a combination of nucleotide sequences: SEQ ID NOS:12 and 13,SEQ ID NOS:14 and 15, SEQ ID NOS:12 and 16, SEQ ID NOS:17 and 18, SEQ IDNOS:19 and 13, or SEQ ID NOS:22 and 23.

Using the chromosomal DNA as a template, PCR is carried out with DNAThermal Cycler (manufactured by Perkin Elmer Instruments, Inc. Japan)using the primers; TaKaRa LA-PCR™ Kit Ver. 2 (manufactured by TAKARASHUZO CO., LTD.) or Expand™ High-Fidelity PCR System (manufactured byBoehringer Manheim K.K.)

In a reaction condition for PCR, PCR is carried out by 30 cycles, in thecase of amplifying a DNA fragment of 2 kb or less, one cycle consistingof reaction at 94° C. for 30 seconds, 55° C. for 30 seconds to 1 minute,and 72° C. for 2 minutes; in the case of amplifying a DNA fragment ofmore than 2 kb, one cycle consisting of reaction at 98° C. for 20seconds, and 68° C. for 3 minutes; then followed by the reaction at 72°C. for 7 minutes.

The amplified DNA fragments are cut at sites the same as the restrictionenzyme sites added to the above primers, and are fractionated andcollected by using agarose gel electrophoresis, sucrose density-gradientcentrifugation and the like.

For cloning the amplified DNA obtained above, an appropriate cloningvector is digested with restriction enzymes creating the cohensive endswhich are able to ligate with the amplified DNA fragment. Using arecombinant DNA obtained by ligating the above amplified DNA with thecloning vector, Escherichia coli, e.g., E coli DH5 α (available fromTOYOBO CO., LTD.) is transformed.

As a cloning vector for cloning the amplified DNA, any cloning vectorsincluding phage vectors and plasmic vectors, which can autonomouslyreplicate in E coli K12, can be used. Expression vectors for E. coli canbe used as cloning vectors. Concrete examples of the cloning vectorsinclude ZAP Express [manufactured by Stratagene, Strategies, 5, 58(1992)], pBluescript II SK(+)[Nucleic Acids Research, 17, 9494 (1989)],Lambda ZAP II (manufactured by Stratagene), λgt10, λgt11 (DNA Cloning, APractical Approach, 1, 49 (1985)), λTriplEx (manufactured by Clonetec),λExCell (manufactured by Pharmacia), pT7T318U (manufactured byPharmacia), pcD2 [H. Okayama and P. Berg; Mol. Cell. Biol, 3, 280(1983)], pMW218 (manufactured by WAKO PURE CHEMICAL INDUSTRIES., LTD),pUC118 (manufactured by TAKARA SHUZO CO., LTD.), pEG400 [J. Bac., 172,2392 (1990)], and pQE-30 (manufactured by Qiagen. Inc).

A plasmid DNA containing a DNA of interest can be obtained from theresultant transformant according to standard techniques, such as thosedescribed in Molecular Cloning, Second Edition, Current Protocols inMolecular Biology, Supplement 1 to 38, John Wiley & Sons (1987-1997),DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition,Oxford University Press (1995).

A plasmid DNA containing a DNA encoding a protein having activity tocatalyze the reaction to produce 1-deoxy-D-xylulose 5-phosphate frompyruvic acid and glyceraldehyde 3-phosphate, a DNA encoding farnesylpyrophosphate synthase, a DNA encoding a protein having an amino acidsequence of SEQ ID NO:3, a DNA encoding a protein having an amino acidsequence of SEQ ID NO:4 or the like; and a plasmid DNA containing one ormore DNAs above, can be obtained by the above methods.

Such plasmids include plasmid pADO-1 that contains all of the DNA above,plasmid pDXS-1 or pQEDXS-1 that contains a DNA having a nucleotidesequence of SEQ ID NO:6, plasmid pISP-1 that contains a DNA having anucleotide sequence of SEQ ID NO:7, plasmid pXSE-1 that contains a DNAhaving a nucleotide sequence of SEQ ID NO:8, and plasmid pTFE-1 thatcontains a DNA having a nucleotide sequence of SEQ ID NO:9.

Using the nucleotide sequences of DNA fragments derived from E. coli,which have been inserted into these plasmids, homologues of the DNA canbe obtained from other prokaryotes, such as microorganisms belonging tothe genus Rhodobacter, in the same manner as described above.

(2) Cloning of DNA Encoding a Protein Having Activity to ComplementMethylerythritol-Requiring Mutant of E. coli (Gene ComplementingMethylerythritol-Requiring Mutant)

Construction of E. coli Methylerythritol-Requiring Mutant

Escherichia coli, such as E. coli W3110 (ATCC14948), is culturedaccording to standard techniques.

After culturing, cells are recovered from the obtained culture bycentrifugation.

The obtained cells are washed with an appropriate buffer agent, such as0.05 mol/l Tris-maleate buffer (pH 6.0). Then the cells are suspended inthe same buffer such that the cell density is 10⁴ to 10¹⁰ cells/ml.

Mutagenesis is carried out by standard techniques using the suspension.In such a standard technique, for example, NTG is added to thesuspension to a final concentration of 600 mg/l, and then the mixture ismaintained for 20 minutes at room temperature.

This suspension after mutagenesis is spread on minimal agar mediumsupplemented with 0.05 to 0.5% methylerythritol and cultured.

An example of minimal agar medium is M9 medium (Molecular Cloning,Second Edition) supplemented with agar.

Methylerythritol that is chemically synthesized according to the methoddescribed in Tetrahedron Letters, 38, 35, 6184 (1997) maybe used.

Colonies grown after culturing are replicated on minimal agar media andminimal agar media each containing 0.05 to 0.5% methylerythritol. Themutant of interest, which requires methylerythritol to grow, isselected. That is, a strain capable of growing on minimal agar mediacontaining methylerythritol but not on minimal agar media lackingmethylerythritol is selected.

Strain ME 7 is an example of the resultant methylerythritol-requiringmutant obtained by the above manipulations.

3 Cloning of the Gene Complementing Methylerythritol-Requiring Nature

Echerichia coli, such as E. coli W3110 (ATCC14948), is inoculated intoculture media, e.g., LB liquid medium, then cultured to the logarithmicgrowth phase by standard techniques.

Cells are collected from the resultant culture by centrifugation.

Chromosomal DNA is isolated and purified from the obtained cellsaccording to standard techniques, such as those described in MolecularCloning, Second Edition. The chromosomal DNA obtained by the methoddescribed in (1) above can be used as isolated and purified chromosomalDNA.

An appropriate amount of the chromosomal DNA is partially digested withan appropriate restriction enzyme, such as Sau 3 A I. The digested DNAfragments are fractionated by according to standard techniques, such assucrose density-gradient centrifugation (26,000 rpm, 20° C., 20 hr).

The DNA fragments obtained by the above fractionation, 4 to 6 kb each,are ligated to a vector, e.g., pMW118 (Nippon Gene), which has beendigested with an appropriate restriction enzyme to construct achromosomal DNA library.

The methylerythritol-requiring mutant isolated in

above, such as the strain ME 7, is transformed using the ligated DNAaccording to standard techniques, e.g., those described in MolecularCloning, Second Edition.

The resulting transformants are spread on minimal agar mediasupplemented with a drug corresponding to a drug-resistant gene carriedby the vector, such as M9 agar medium containing 100 μg/l of ampicillin,then cultured overnight at 37° C.

Thus, transformants that have recovered their methylerythritolrequirement can be selected by the method above.

Plasmids are extracted from the resultant transformants by standardtechniques. Examples of a plasmid that can allow the transformants torecover their methylerythritol requirement are pMEW73 and pQEDXR.

The nucleotide sequence of the DNA integrated into the plasmid issequenced.

An example of such a nucleotide sequence is a sequence containing anuclectide sequence for yaeM gene of SEQ ID NO:10. Using the informationon the nucleotide sequence for yaeM gene, homologues of yaeM gene can beobtained from other prokaryotes or plants in the same manner asdescribed above.

II. Production of Proteins Having Activity to Improve Efficiency in theBiosynthesis of Isoprenoid Compounds

To express the resulting DNA in a host cell, the DNA fragment ofinterest is digested with restriction enzymes or deoxyribonucleases intoone with a proper length containing the gene. Next the fragment isinserted into a downstream of a promoter region in an expression vector.Then the expression vector is introduced into a host cell appropriatefor the expression vector.

Any host cell that can express the gene of interest can be used.Examples of the host cell include bacteria belonging to the generaEscherichia, Serratia, Corynebacterium, Brevibacterium, Pseudomonas,Bacillus, Microbacterium and the like, yeasts belonging to the generaKluyveromyces, Saccharomyces, Schizosaccharomyces, Trichosporon,Schwanniomyces, and the like, animal cells, and insect cells.

Expression vectors used herein can autonomously replicate in the hostcell above or be integrated into a chromosomal DNA, and contain apromoter at the position to which the DNA of interest as described abovecan be transcribed.

When a bacterium is used as a host cell, a preferable expression vectorfor expression of the DNA above can autonomously replicate in thebacterium and is a recombinant vector comprising a promoter, ribosomebinding sequence, the DNA above and a transcription terminationsequence. The expression vector may contain a gene to regulate apromoter.

Examples of the expression vector include pBTtp2, pBTac1, pBTac2 (all ofthem are available from Boehringer Manheim K.K.), pKK233-2 (Pharmacia),pSE280 (Invitrogen), pGEMEX-1 (Promega), pQE-8 (Qiagen. Inc), pQE-30(Qiagen. Inc), pKYP10 (Japanese Patent Laid Open Publication No,58-110600), pKYP200 (Agricultural Biological Chemistry, 48, 669, 1984),pLSA1 (Agric. Biol. Chem, 53, 277, 1989), pGEL1 (Proc. Natl. Acad. Sci.USA, 82, 4306, 1985), pBluescriptII SK+, pBluescriptII SK (−)(Stratagene), pTrS30 (FERM BP-5407), pTrS32 (FERM BP-5408), pGEX(Pharmacia), pET-3 (Novagen), pTerm2 (U.S. Pat. Nos. 4,686,191,4,939,094, 5,160,735), pSupex, pUB110, pTP5, pC194, pUC18 (Gene, 33,103, 1985), pUC19 (Gene, 33, 103, 1985), pSTV28 (TAKARA SHUZO CO.,LTD.), pSTV29 (TAKARA SHUZO CO., LTD.), pUC118 (TAKARA SHUZO CO., LTD.),pPA1 (Japanese Patent Laid Open Publication No. 63-233798), pEG400 (J.Bacteriol., 172, 2392, 1990), and pQE-30 (Qiagen. Inc).

Any promoter that can function in a host cell may be used. Examples ofsuch a promoter include promoters derived from Escherichia coli orphages, such as trp promoter (P trp), lac promoter (P lac), P_(L)promoter, P_(R) promoter, P_(SE) promoter, SP01 promoter, SP02 promoter,and penP promoter. Furthermore, P trp×2 promoter that is formed byjoining two P trp in series, and tac promoter, letI promoter, and lacT7promoter, those artificially designed and modified, can be used.

Any ribosome binding sequence that can function in a host cell can beused. A preferable plasmid has a distance between Shine-Dalgarnosequence and a starting codon appropriately adjusted, of for example 6to 18 bases long.

A transcription termination sequence is not always required forexpression of the DNA of interest. Preferably, a transcriptiontermination sequence is arranged immediately followed by a structuralgene.

Examples of the host cell used herein include microorganisms belongingto the genera Escherichia, Corynebacterium, Brevibacterium, Bacillus,Microbacterium, Serratia, Pseudomonas, Agrobacterium, Alicyclobacillus,Anabaena, Anacystis, Arthrobacter, Azobacter, Chromatium, Erwinia,Methylobacterium, Phormidium, Rhodobacter, Rhodopseudomonas,Rhodospirillum, Scenedesmun, Streptomyces, Synnecoccus, and Zymomonas.Preferable host cells include microorganisms belonging to the generaEscherichia, Corynebacterium, Brevibacterium, Bacillus, Pseudomonas,Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Arthrobacter,Azobacter, Chromatium, Erwinia, Methylobacterium, Phormidium,Rhodobacter, Rhodopseudomonas, Rhodospirillum, Scenedesmun,Streptomyces, Synnecoccus and Zymomonas.

More specific examples of the host cell include Escherichia coliXL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichiacoli DH5a, Escherichia coli MC1000, Escherichia coli KY3276, Escherichiacoli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichiacoli No. 49, Escherichia coli W3110, Escherichia coli NY49, Escherichiacoli MP347, Escherichia coli NM522, Bacillus subtilis, Bacillusamyloliquefacines, Brevibacterium ammoniagenes, Brevibacteriumimmariophilum ATCC14068, Brevibacterium saccharolyticum ATCC14066,Brevibacterium flavum ATCC14067, Brevibacterium lactofermentumATCC13869, Corynebacterium glutamicum ATCC13032, Corynebacteriumglutamicum ATCC14297, Corynebacterium acetoacidophilum ATCC13870,Microbacterium ammoniaphilum ATCC15354, Serratia ficaria, Serratiafonticola, Serratia liquefaciens, Serratia marcescens, Pseudomonas sp.D-0110, Agrobacterium radiobacter, Agrobacterium rhizogenes,Agrobacterium rubi, Anabaena cylindrica, Anabaena doliolum, Anbaenaflos-aquae, Arthrobacter aurescens, Arthrobacter citreus, Arthrobacterglobformis, Arthrobacter hydrocarboglutamicus, Arthrobacter mysorens,Arthrobacter nicotianae, Arthrobacter paraffineus, Arthrobacterprotophormiae, Arthrobacter roseoparaffinus, Arthrobacter sulfureus,Arthrobacter ureafaciens, Chromatium buderi, Chromatium tepidum,Chromatium vinosum, Chromatium warmingii, Chromatium fluviatile, Erwiniauredovora, Erwinia carotovora, Erwnia ananas, Erwinia herbicola, Erwiniapunctata, Erwinia terreus, Mehylobacterium rhodesianum, Methylobacteriumextorquens, Phomidium sp. ATCC29409, Rhodobacter capsulatus, Rhodobactersphaeroides, Rhodopseudomonas blastica, Rhodopseudomonas marina,Rhodopseudomonas palustris, Rhodospirillum rubrum, Rhodospirillumsalexigens, Rhodospirillum salinarum, Streptomyces ambofaciens,Streptomyces aureofaciens, Streptomyces aureus, Streptomycesfungicidicus, Streptomyces griseochromogenes, Streptomyces griseus,Streptomyces lividans, Streptomyces olivogriseus, Streptomyces rameus,Streptomyces tanashiensis, Streptomyces vinaceus, and Zymomonas mobilis.

Any method to introduce a recombinant vector into the host cell asdescribed above may be used. Examples of such a method include a methodusing calcium ions (Proc. Natl. Acad. Sci. USA, 69, 2110, 1972),protoplast method (Japanese Patent Laid Open Publication No.63-2483942), or methods described in Gene, 17, 107 (1982) or Molecular &General Genetics, 168, 111 (1979).

When yeast is used as a host cell, expression vectors are, for example,YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, andpHS15.

Any promoter that can function in yeast can be used. Examples of such apromoter include PH05 promoter, PGK promoter, GAP promoter, ADHpromoter, gal 1 promoter, gal 10 promoter, heat shock protein promoter,MF α1 promoter, and CUP1 promoter.

Host cells used herein include Saccharomyces cerevisae,Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans,and Schwanniomyces alluvius.

Any method to introduce a recombinant vector, that is, to introduce DNAinto yeast may be used. Examples of such methods include Electroporation(Methods. Enzymol., 194, 182, 1990), Spheroplast method (Proc. Natl.Acad. Sci. USA, 75, 1929 (1978)), lithium acetate method (J. Bacteriol.,153, 163 (1983)), and methods described in Proc. Natl. Acad. Sci. USA,75, 1929 (1978).

When an animal cell is used as a host cell, expression vectors are, forexample, pcDNAI, pcDM8 (Funakoshi Co., Ltd), pAGE107 [Japanese PatentLaid Open Publication No. 3-22979; Cytotechnology, 3, 133 (1990)],pAS3-3 [Japanese Patent Laid Open Publication No. 2-227075, pCDM8(Nature, 329, 840 (1987)), pcDNAI/Amp (Invitrogen), pREP4 (Invitrogen),pAGE103 [J. Biochem. 101, 1307 (1987)], and pAGE210.

Any promoter that can function in an animal cell may be used. Examplesof such promoters include a promoter for IE (immediate early) gene ofcytomegalovirus (human CMV), SV40 initial promoter, retrovirus promoter,metallothionein promoter, heat shock promoter, and SR α promoter.Moreover, an enhancer of human CMV IE gene may be used together with apromoter.

Host cells used herein are, for example, Namalwa cells, HBT5637(Japanese Patent Laid Open Publication No. 63-299), COS1 cells, COS7cells, and CHO cells.

Any method to introduce a recombinant vector into an animal cell, thatis, to introduce DNA into an animal cell may be used. Examples of suchmethods include Electroporation [Cytotechnology, 3, 133 (1990)], calciumphosphate method (Japanese Patent Laid Open Publication No. 2-227075),lipofection [Proc. Natl. Acad. Sci., USA, 84, 7413 (1987)], and methodsdescribed in Virology, 52, 456 (1973). Recovery and culture of thetransformant can be carried out according to methods described inJapanese Patent Laid Open Publication No. 2-227075 and Japanese PatentLaid Open Publication No. 2-257891.

When an insect cell is used as a host cell, proteins can be expressedaccording to methods described in, such as Baculovirus ExpressionVectors, A Laboratory Manual, Current Protocols in Molecular BiologySupplement 1-38 (1987-1997), and Bio/Technology, 6, 47 (1988).

That is, a vector for introducing a recombinant gene and Baculovirus areco-transduced into an insect cell to obtain a recombinant virus in theculture supernatant of the insect cell. Then an insect cell is infectedwith the recombinant virus, resulting in expression of the protein ofinterest.

Examples of the vectors to transfer genes include pVL1392, pVL1393,pBlueBacIII (all of which are manufactured by Invitrogen).

Baculoviruses used herein are, for example, Autographa califomicanuclear polyhedrosis virus that infects Barathra insects.

Examples of the insect cells include ovarian cells of Spodopterafrugiperda, Sf9, and Sf21 (Baculovirus Expression Vectors, A LaboratoryManual (W. H. Freeman and Company, New York, 1992), and of Trichoplusiani, High 5 (Invitrogen).

Methods of co-transduction of the vector for transferring therecombinant gene and the Baculovirus into an insect cell to prepare arecombinant virus include calcium phosphate transfection (JapanesePatent Laid Open Publication No. 2-227075) and, lipofection [Proc. Natl.Acad. Sci. USA, 84, 7413 (1987)].

Methods for expressing genes include secretory production, and fusionprotein expression according to the techniques shown in MolecularConing, Second Edition, in addition to direct expression.

When the gene is expressed in yeasts, animal cells, or insect cells, aprotein to which sugar or a sugar chain is added, can be obtained.

Proteins having activity to improve efficiency in the biosynthesis ofisoprenoid compounds can be produced by culturing a transformantcontaining a recombinant DNA to which the above DNA has been introducedin a medium, allowing the transformant to produce and accumulateproteins having activity to improve efficiency in the biosynthesis ofisoprenoid compounds in the culture, then collecting the proteins fromthe culture.

The transformants for producing proteins with activity to improveefficiency in the biosynthesis of isoprenoid compounds of the presentinvention, can be cultured by standard techniques to culture a hostcell.

When the transformant of this invention is prokaryote such asEscherichia coli or eukaryote such as yeast, a medium for culturing suchtransformants contains a carbon source, a nitrogen source, and inorganicsalts, which the microorganisms can assimilate, and allows thetransformant to grow efficiently. Ether natural media or synthetic mediacan be used if they satisfy the above conditions.

Any carbon source assimilable by the microorganisms may be used. Suchcarbon sources include glucose, fructose, sucrose, and molassescontaining them, carbohydrates e.g., starch or hydrolysates of starch,organic acids e.g., acetic acid and propionic acid, and alcohols e.g.,ethanol and propanol.

Examples of nitrogen sources include ammonia, salts of inorganic acidsor organic acids, e.g., ammonium chloride, ammonium sulfate, ammoniumacetate, and ammonium phosphate, other nitrogen-containing compounds,peptone, meat extract, yeast extract, corn steep liquor, caseinhydrolysates, soybean meal and soybean meal hydrolysate, variousfermentation microorganic cells or their digests.

Examples of inorganic salts include potassium primary phosphate,potassium secondary phosphate, magnesium phosphate, magnesium sulfate,sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, andcalcium carbonate.

Culturing is carried out by shaking culture or submergedaeration-agitation culture are carried out under aerobic conditions. Thepreferable culture temperature ranges from 15 to 40° C. The preferableculture period ranges from 16 hours to 7 days. The pH is kept within arange from 3.0 to 9.0 while culturing. The pH is adjusted usinginorganic or organic acid, alkaline solutions, urea, calcium carbonate,ammonia or the like.

If necessary, an antibiotics e.g., ampicillin or tetracycline may beadded to the media while culturing.

When microorganisms transformed with the expression vectors usinginducible promoters are cultured, inducers may be added to the media ifnecessary. For example, isopropyl-β-D-thiogalactopyranoside (IPTG) orthe like may be added to the media when microorganisms transformed withthe expression vectors containing lac promoter are cultured;indoleacrylic acid (IAA) or the like may be added when microorganismstransformed with the expression vectors containing trp promoter arecultured.

The media for culturing a transformant obtained by using an animal cellas a host cell include a generally used RPMI1640 medium [The Journal ofthe American Medical Association, 199, 519 (1967)], Eagle's MEM medium[Science, 122, 501 (1952)], DMEM medium [Virology, 8, 396 (1959)], 199medium [Proceeding of the Society for the Biological Medicine, 73, 1(1950)] or those to which fetal calf serum or the like is added.

Normally, the transformant is cultured in the presence of 5% CO₂ for 1to 7 days at pH 6 to 8 and 30 to 40° C.

If necessary, antibiotics e.g., kanamycin and penicillin may be added tothe medium while culturing.

Examples of media to culture a transformant obtained by using an insectcell as a host cell include a generally used TNM-FH medium (Pharmingen),Sf-900 II SFM medium (GIBCO BRL), ExCell400, ExCell405 (bothmanufactured by JRH Biosciences), Grace's Insect Medium (Grace, T.C.C.,Nature, 195, 788 (1962)).

The transformant is generally cultured for 1 to 5 days at pH 6 to 7 andat 25° C. to 30° C.

If necessary, antibiotics e.g., gentamycin may be added to the mediumwhile culturing.

Proteins having activity to improve efficiency in the biosynthesis ofisoprenoid compounds of this invention can be isolated and purified fromthe culture of the transformant of this invention by standard isolationand purification techniques for a enzyme.

For example, when the protein of this invention is expressed in asoluble form within the cell, after the culture is completed the cellsare recovered by centrifugation, suspended in aqueous buffer, thendisrupted using an ultrasonicator, french press, Manton Gaulinhomogenizer, Dyno-Mill, or the like, thereby obtaining cell-freeextracts. The cell-free extract is separated by centrifugation to obtainthe supernatant. The purified sample can be obtained from thesupernatant by one of or a combination of standard techniques forisolating and purifying enzymes. Such techniques include a solventextracting technique, salting out technique using ammonium sulfate,desalting technique, precipitation technique using organic solvents,anion exchange chromatography using resins such as diethylaminoethyl(DEAE)-Sepharose, and DIAION HPA-75 (Mitsubishi Chemical Corp.), cationexchange chromatography using resins e.g., S-Sepharose FF (Pharmacia),hydrophobic chromatography using resins e.g., butylsepharose,phenylsepharose, gel filtration using molecular sieve, affinitychromatography, chromatofocusing, and electrophoresis such asisoelectric focusing.

When the proteins that form inclusion bodies are expressed in the cells,the cells are recovered, disrupted, and separated by centrifugation,thereby obtaining precipitated fractions. From the resultingprecipitated fractions, the protein is recovered by standard techniques,and then the insoluble protein is solubilized using a protein denaturingagent. The solubilized solution is diluted or dialyzed to an extent thatthe solution contains no protein denaturing agent or that theconcentration of protein denaturing agent does not denature protein,thereby allowing the protein to form a normal three-dimensionalstructure. Then the purified sample can be obtained by the sametechniques for isolation and purification as described above.

When the protein of this invention or its derivative, such as asugar-modified protein, is secreted outside the cell, the protein or itsderivative, such as a sugar chain adduct, can be recovered from theculture supernatant. That is, the culture is treated by centrifugationand the like as described above so as to obtain soluble fractions. Fromthe soluble fractions, the purified sample can be obtained using thetechniques for isolation and purification as described above.

The resulting protein as described above is, for example a proteinhaving an amino acid sequence selected from amino acid sequences of SEQID NOS:1 to 5.

Moreover, the protein expressed by the method above can be chemicallysynthesized by techniques including Fmoc method(fluorenylmethyloxycarbonyl method), tBoc method (t-butyloxycarbonylmethod). Further, the protein can be synthesized by using a peptidesynthesizer of Souwa Boeki K.K. (Advanced ChemTech, U.S.A), Perkin-ElmerJapan (Perkin-Elmer, U.S.A), Pharmacia BioTech (Pharmacia BioTech,Sweden), ALOKA CO., LTD. (Protein Technology Instrument), KURABOINDUSTRIES LTD. (Synthecell-Vega, U.S.A), PerSeptive Limited., Japan(PerSeptive, U.S.A), or SHIMADZU CORP.

III. Production of Isoprenoid Compound

Isoprenoid compounds can be produced by culturing the transformantsobtained as described in II above according to the method of II above,allowing the transformants to produce and accumulate isoprenoidcompounds in the culture, then recovering the isoprenoid compounds fromthe culture.

The above culture can yield isoprenoid compounds, such as ubiquinone,vitamin K₂, and carotenoids. Specific examples of isoprenoid compoundsinclude ubiquinone-8 and menaquinone-8 produced using microorganismsbelonging to the genus Escherichia as a transformant, ubiquinone-10produced using those belonging to the genus Rhodobacter, vitamin K₂produced using those belonging to the genus Arthrobacter as atransformant, astaxanthin produced using those belonging to the genusAgrobacterium as a transformant, and lycopene, β-carotene, andzeaxanthin produced using those belonging to the genus Erwinia as atransformant.

After the culture is completed, in order to isolate and purifyisoprenoid compounds, isoprenoid compounds are extracted by adding anappropriate solvent to the culture, the precipitate is removed by e.g.,centrifugation, and then the product is subjected to variouschromatography.

IV. Screening a Substance Inhibiting Enzymatic Activity onNon-Mevalonate Pathway

(1) Determination of Enzymatic Activity on Non-Mevalonate Pathway

The enzymatic activity on non-mevalonate pathway can be determinedaccording to normal methods for determining enzymatic activity.

The pH of the buffer used as a reaction solution to determine activityshould be within a range that does not inhibit the enzymatic activity ofinterest. A preferable pH range includes the optimal pH.

For example, a buffer at pH 5 to 10, preferably 6 to 9 is used for1-deoxy-D-xylulose 5-phosphate reductoisomerase.

Any buffer can be used herein so far as it does not inhibit theenzymatic activity and can be adjusted to the pH above. Examples of sucha buffer include Tris-hydrochloric acid buffer phosphate buffer, boratebuffer, HEPES buffer, MOPS buffer, and bicarbonate buffer. For example,Tris-hydrochloric acid buffer can preferably be used for1-deoxy-D-xylulose 5-phosphate reductoisomerase.

A buffer of any concentration may be employed so far as it does notinhibit the enzymatic activity. The preferable concentration ranges from1 mol/l to 1 mol/l.

When the enzyme of interest requires a coenzyme, a coenzyme is added tothe reaction solution. For example, NADPH, NADH or other electron donorscan be used as a coenzyme for 1-deoxy-D-xylulose 5-phosphatereductoisomerase. A preferable coenzyme is NADPH.

Any concentration of the coenzyme to be added can be employed so far asit does not inhibit reaction. Such a concentration preferably rangesfrom 0.01 mol/l to 100 mol/l, more preferably, 0.1 mol/l to 10 mol/l.

Metal ions may be added to a reaction solution if necessary. Any metalion can be added so far as it does not inhibit reaction. Preferablemetal ions include Co²⁺, Mg²⁺, and Mn²⁺.

Metal ions may be added as metallic salts. For example, a chloride, asulfate, a carbonate, and a phosphate can be added.

Any concentration of the metal ion to be added can be employed so far asit does not inhibit reaction. A preferable concentration ranges from 0mol/l to 100 mol/l, more preferably, 0.1 mol/l to 10 mol/l.

The substrate of the enzyme of interest is added to the reactionsolution. For example, 1-deoxy-D-xylulose 5-phosphate is added for1-deoxy-D-xylulose 5-phosphate reductoisomerase.

Any concentration of the substrate may be employed so far as it does notinhibit reaction. The preferable concentration ranges from 0.01 mol/l to0.2 mol/l in the reaction solution.

The enzyme concentration used in reaction is not specifically limited.Normally, the concentration ranges from 0.01 mg/ml to 100 mg/ml.

An enzyme used herein is not necessarily purified into a singlesubstance. It may contain contaminative proteins. In the search asdescribed in (2) below, cellular extracts containing 1-deoxy-D-xylulose5-phosphate reductoisomerase activity or cells having the same activitycan be used.

Any reaction temperature may be employed so far as it does not inhibitenzymatic activity. A preferable temperature range includes the optimaltemperature. That is, the reaction temperature ranges from 10° C. to 60°C., more preferably, 30° C. to 40° C.

Activity can be detected by a method for measuring a decrease insubstrates accompanying the reaction or an increase in reaction productsas the reaction proceeds.

Such a method is a method wherein the substance of interest is separatedand quantitatively determined by e.g, high performance liquidchromatography (HPLC) if necessary. When NADH or NADPH increases ordecreases as the reaction proceeds, activity can directly be determinedby measuring the absorbance at 340 nm of the reaction solution. Forexample, the activity of 1-deoxy-D-xylulose 5-phosphate reductoisomerasecan be detected by measuring a decrease in the absorbance at 340 nmusing a spectrophotometer to determine NADPH quantity that decreases asthe reaction proceeds.

(2) Screening a Substance Inhibiting Enzymatic Activity on theNon-Mevalonate Pathway

A substance inhibiting enzymatic activity on the non-mevalonate pathwaycan be screened for by adding the substance to be screened for to theenzymatic activity measurement system as described in (1) above,allowing the mixture to react similarly, and then screening a substancethat suppresses the amount of the substrates decreased in comparison toa case when no such substance is added; or a substance that suppressesthe yield of the reaction product.

Screening methods include a method wherein the decrease in the amount ofsubstrates or the increase in the amount of reaction products is tracedwith time; or a method where after the reaction has proceeded for acertain period the decrease in the amount of substrates or the increasein the amount of reaction products is measured.

In the method wherein the decrease in the amount of substrates or theincrease in the amount of reaction products is traced with time, theamount is measured preferably at 15 seconds to 20 minutes intervals,mere preferably at 1 to 3 minutes intervals during reaction.

To measure the decrease in the amount of substrates or the increase inthe amount of reaction products after reaction has proceeded for acertain period, the reaction period is preferably 10 minutes to 1 day,more preferably, 30 minutes to 2 hours.

A substance inhibiting the enzymatic activity on the non-mevalonatepathway inhibits the growth of microorganisms and plants that possessthe non-mevalonate pathway. The inventors have first found the fact thatthis substance inhibits the growth of the microorganisms and plants.

The non-mevalonate pathway is present in microorganisms and plants, butabsent in animals and humans. Therefore, the substance inhibiting theenzymatic activity on the non-mevalonate pathway but not affecting humanand animals can be obtained by the above described screening method.

This substance can be an effective antibiotic or herbicide.

This specification includes part or all of the contents as disclosed inthe specification and/or drawings of Japanese Patent Application Nos.10-103101, 10-221910 and 11-035739, which are priority documents of thepresent application.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the effect of reaction temperature on 1-deoxy-D-xylulose5-phosphate reductoisomerase activity.

FIG. 2 shows the effect of the pH of the reaction solution on1-deoxy-D-xylulose 5-phosphate reductoisomerase activity. Enzymaticactivity measured at various pH in 100 mol/l Tris-hydrochloric acidbuffer are shown. Activity is shown as a relative activity when activityat pH 8.0 is considered as 100%.

FIG. 3 shows a method for disrupting yaeM gene on a chromosome usinghomologous recombination.

FIG. 4 shows the effect of fosmidomycin on 1-deoxy-D-xylulose5-phosphate reductoisomerase.

BEST MODE FOR CARRYING OUT THE INVENTION

The invention will now be described by way of examples, but shall not belimited thereto. Unless otherwise specified, gene recombination shown inthe examples was carried out according to techniques described inMolecular Cloning, Second Edition (hereinafter referred to as thestandard techniques).

EXAMPLE 1 Cloning of DNA Encoding Proteins Involved in the Biosynthesisof Isoprenoid Compounds

(1) Cloning of DNA Encoding Proteins Involved in the Biosynthesis ofIsoprenoid Compounds Using the Nucleotide Sequence of E. coli DXS Gene

One platinum loop of E. coli XL1-Blue (purchased from TOYOBO) wasinoculated into 10 ml of LB liquid medium, then cultured overnight at37° C.

After culturing, cells were collected by centrifugation from theresultant culture.

Chromosomal DNA was isolated and purified from the cells according tothe standard techniques.

Sense and antisense primers, each having BamHI and EcoR I restrictionenzyme sites at their 5′-ends and consisting of nucleotide sequencepairs of SEQ ID NOS:12 and 13, 14 and 15, 12 and 16, 17 and 18, and 19and 13; and sense and antisense primers, each having BamHI restrictionenzyme site at their 5′-ends and consisting of a nucleotide sequencepair of SEQ ID NO:22 and 23; were synthesized using a DNA synthesizer.

PCR was carried out with a DNA Thermal Cycler (Perkin Elmer Instruments,Inc. Japan) using these primers, chromosomal DNA as a template, and aTaKaRa La-PCR™ Kit Ver. 2 (TAKARA SHUZO CO., LTD.), Expand™High-Fidelity PCR System (Boehringer Manheim K.K.) or a Taq DNApolymerase (Boehringer).

PCR was carried out for 30 cycles. In the case of amplifying a DNAfragment of 2 kb or less, one cycle consisting of reaction at 94° C. for30 seconds, 55° C. for 30 seconds to 1 minute, and 72° C. for 2 minutes;in the case of amplifying a DNA fragment of more than 2 kb, one cycleconsisting of reaction at 98° C. for 20 seconds, and 68° C. for 3minutes; then followed by the reaction at 72° C. for 7 minutes.

Among the DNA fragments amplified by PCR, DNA fragments amplified usingsense and antisense primers, each having BamH I and EcoR I restrictionenzyme sites at their 5′-ends, were digested with restriction enzymesBamH I and EcoR I; DNA fragments amplified using sense and antisenseprimers, each having BamH I restriction enzyme site at their 5′-ends,were digested with restriction enzyme BamH I.

After the digestion, these DNA fragments treated with the restrictionenzymes were subjected to agarose gel electrophoresis and recovered BamHI and EcoR I-treated DNA fragments and BamH I-treated DNA fragments.

A broad host range vector pEG 400 containing lac promoter [J. Bac., 172,2392 (1990)] was digested with restriction enzymes BamH I and EcoR I,subjected to agarose gel electrophoresis and recovered BamH I and EcoRI-treated pEG 400 fragments.

pUC118 (TAKARA SHUZO CO., LTD.) was digested with a restriction enzymeBamH I, then subjected to agarose gel electrophoresis and recovered BamHI-treated pUC 118 fragments.

Each of the resultant BamH I and EcoR I-treated DNA fragments was mixedwith BamH I and EcoR I-treated pEG 400 fragments, then the mixture wasallowed to precipitate with ethanol. The obtained DNA precipitate wasdissolved in 5 μl of distilled water for ligation reaction to occur,thereby obtaining each recombinant DNA.

Using the resultant recombinant DNA, E. coli (purchased from TOYOBO) DH5α was transformed according to the standard techniques. Then thetransformant was spread on LB agar medium containing 100 μg/ml ofspectinomycin, then cultured overnight at 37° C.

Some colonies of the transformant resistant to spectinomycin werecultured in 10 ml of LB liquid medium containing 100 μg/ml ofspectinomycin with shaking for 16 hours at 37° C.

The resulting culture was centrifuged, so that cells were collected.

Plasmids were isolated from the cells according to the standardtechniques.

To confirm that the isolated plasmids contained the DNA fragment ofinterest, the plasmids were cleaved with various restriction enzymes toexamine their structures and their nucleotide sequences were sequenced.

A plasmid containing a DNA with a nucleotide sequence of SEQ ID NO:6,DNA with a nucleotide sequence of SEQ ID NO:7, DNA with a nucleotidesequence of SEQ ID NO:8, and DNA with a nucleotide sequence of SEQ IDNO:9 was named pADO-1. A plasmid containing a DNA with a nucleotidesequence of SEQ ID NO:6 was named pDXS-1. A plasmid containing a DNAwith a nucleotide sequence of SEQ ID NO:7 was named pISP-1. A plasmidcontaining a DNA with a nucleotide sequence of SEQ ID NO:9 was namedpTFE-1.

The above BamH I-treated DNA fragments and BamH I-treated pUC118fragments were mixed, then the mixture was allowed to precipitate withethanol. The resulting DNA precipitate was dissolved in 5 μl ofdistilled water for ligation reaction to occur to obtain recombinantDNA. Escherichia coli was transformed using the recombinant DNA in thesame manner as described above, then plasmids were isolated from thetransformants.

To confirm the isolated plasmids contain the DNA fragments of interest,the plasmids were cleaved with various restriction enzymes to examinetheir structures and their nucleotide sequences were sequenced in thesame manner as described above.

These plasmids were digested with BamH I. The DNA fragments of interestwere recovered in the same manner as described above, then sub-clonedinto an expression vector pQE30 (Qiagen, Inc).

The plasmid obtained by the sub-cloning above and having a nucleotidesequence of SEQ ID NO:6 was named pQEDXS-1.

(2) Cloning of the Gene Complementing Methylerythritol-Requiring Nature

Selection of Methylerythritol-Requiring Mutant of Escherichia coli

E. coli W3110 (ATCC 14948) was inoculated into LB liquid medium andcultured to its logarithmic growth phase.

After culturing, cells were recovered from the resulting culture bycentrifugation.

The cells were washed with 0.05 mol/l Tris-maleate buffer (pH 6.0), thensuspended in the same buffer to the cell density of 10⁹ cells/ml.

Mutation was induced by adding NTG to the suspension to a fmalconcentration of 600 mg/l, and then the mixture was maintained for 20minutes at room temperature.

These NTG treated cells were spread on M9 minimal agar medium containing0.1% methylerythritol (Molecular Cloning, Second Edition) plate andcultured.

Methylerythritol was chemically synthesized according to the methoddescribed in Tetrahedron Letters, 38, 35, 6184 (1997).

Colonies grown on M9 minimal agar medium containing 0.1%methylerythritol were replicated on M9 minimal agar medium and on M9minimal agar medium containing 0.1% methylerythritol. The mutant ofinterest, a strain requiring methylerythritol to grow, was selected.That is, a strain capable of growing on a minimal agar medium containing0.1% methylerythritol but not on the same lacking methylerythritol wasselected.

The thus obtained methylerythritol-requiring mutant ME7 was used in thefollowing experiments.

Cloning of the Gene Complementing Methylerythritol-Requiring Nature

Escherichia coli W3110 (ATCC14948) was inoculated into LB liquid medium,then cultured to its logarithmic growth phase. Then cells were collectedfrom the resultant culture by centrifugation.

Chromosomal DNA was isolated and purified from the obtained cellsaccording to the standard techniques.

200 μg of the chromosomal DNA was partially digested with a restrictionenzyme, Sau 3AI. The resulting DNA fragments were fractionated bysucrose density-gradient centrifugation (26,000 rpm, 20° C., 20 hr).

The DNA fragments obtained by the above fractionation, 4 to 6 kb each,were ligated to pMW118 vector (Nippon Gene), which had been digestedwith a restriction enzyme BamH I, constructing a genomic DNA library.

Using this genomic DNA library, the strain ME7 isolated in

above was transformed according to the standard techniques.

The resulting transformants were spread on LB agar medium supplementedwith 100 μg/l of ampicillin, then cultured overnight at 37° C.

Plasmids were extracted from each colony that grew on the agar mediumand then the nucleotide sequences were determined.

The plasmids determined its nucleotides sequence had contained thenucleotide sequence of SEQ ID NO:10. These plasmids were named pMEW41and pMEW73.

A plasmid extracted from one strain of the clones having the sequencewas named pMEW73.

The pMEW73 was double-digested with Hind III and Sac I. The resultantHind III and Sac I-treated DNA fragment having a nucleotide sequence ofSEQ ID NO:10 was ligated to multi-cloning sites of broad host rangevector pEG400 [J. Bac., 172, 2392 (1990)], constructing pEGYM1.

The Hind III-Sac I-treated DNA fragment was ligated to the Hind III-SacI site of vector pUC19 (Gene, 33, 103 (1985)), constructing pUCYM-1.

According to the information on the nucleotide sequence of chromosomalDNA of Escherichia coli based on Genbank data base, the DNA fragmentthat had been inserted into the vector was confirmed to contain yaeMgene.

A recombinant vector, which can express yaeM gene sufficiently, wasconstructed by following method with PCR [Science, 230, 1350 (1985)].

A sense primer having a sequence of SEQ ID NO:20 and an antisense primerhaving a sequence of SEQ ID NO:21 were synthesized using a DNAsynthesizer.

A Bam H I restriction enzyme recognition site was added to each 5′-endof the sense and antisense primers.

yaeM gene was amplified by PCR with DNA Thermal Cycler (Perkin ElmerInstruments, Inc. Japan) using chromosomal DNA of E. coli as a template,these primers and Taq DNA polymerase (Boelinnger).

PCR was carried out by 30 cycles, one cycle consisting of reaction at94° C. for 30 seconds, reaction at 55° C. for 30 seconds, and reactionat 72° C. for 2 minutes followed by reaction at 72° C. for 7 minutes.

After the amplified DNA fragments and pUC118 (TAKARA SHUZO CO., LTD.)were digested with a restriction enzyme BamH I, each of the DNAfragments were purified by agarose gel electrophoresis.

Both of these fragments were mixed, then the mixture was allowed toprecipitate with ethanol. The resultant DNA precipitate was dissolved in5 μl of distilled water for ligation reaction to occur, therebyobtaining recombinant DNA.

The recombinant DNA was confirmed to be yaeM gene by determining thenucleotide sequences, then sub-cloned to expression vector pQE30(Qiagen, Inc).

The resulting recombinant DNA was named pQEYM1.

The strain ME7 was transformed using pQEYM1 by standard techniques. Thetransformant was spread on LB agar medium containing 100 μg/ml ofampicillin, then cultured overnight at 37° C.

The transformants were confirmed to form colonies at the same growthrate as wild-type strain, suggesting that yaeM gene complementedmutation in the strain ME7.

EXAMPLE 2 Production of Ubiquinone-8 (CoQ8) Using RecombinantEscherichia coli

(1) E. coli DH5α were transformed using the plasmids pADO-1, pDXS-1, andpXSE-1, those obtained in Example 1 above, and pEG400 as a control,respectively, then E. coli DH5 α/pAD0-1, E. coli DH5 α/pDXS-1, E. coliDH5 α/pXSE-1 and E. coli DH5 α/pEG400 that showed resistance tospectinomycin at a concentration of 100 μg/ml were obtained.

These transformants were inoculated into a test tube containing 10 ml ofLB medium supplemented with thiamine and vitamin B₆, 100 mg/l each, 50mg/l of p-hydroxybenzoic acid, and 100 μg/ml of spectinomycin. Then thetransformants were cultured with shaking for 72 hours at 30° C.

After the culture was completed, each culture was concentrated 10-fold.

To each 300 μl of concentrated culture, 300 μl 2-butanol and 300 μLglass beads were added. Isoprenoid compounds were extracted with thesolvent while disrupting the cells by Multi Beads Shocker MB-200 (YASUIKIKAI) for 5 minutes. Then the 2-butanol layer was collected bycentrifugation.

The amount of CoQ8 produced by the transformants was calculated byQuantitative analysis of the CoQ8 in the butanol layer using highperformance liquid chromatography (LC-10A, SHIMADZU CORP.).

HPLC was carried out using Develosil ODS-HG-5 (NOMURA CHEMICAL K.K.) asa column, and methanol:n-hexane=8:2 solution as a mobile phase at 1ml/min of the flow rate and 275 nm of the measuring wavelength.

Table 1 shows the results. TABLE 1 CoQ8 Production by transformant ofEscherichia coli Cell Amount Amount of CoQ8 Intracellular Transformant(OD660) Produced (mg/L) Content¹ E. coli 5.8 0.63 1.1 DH5 α/pEG400 E.coli 5.5 0.98 1.8 DH5 α/pADO-1 E. coli 5.2 0.85 1.6 DH5 α/pDXS-1 E. coli5.6 0.67 1.2 DHS α/pXSE-1*¹Intracellular content is shown with a value obtained by dividing a10-fold CoQ8 production (mg/L) by a cell amount (OD660).The amount of CoQ8 produced was significantly higher in DH5 α/pADO-1,DH5 α/pDXS-1 and DH5 α/pXSE-1 than in the control strain DH5 α/pEG400.In particular, the highest productivity was shown by DH5 α/pADO-1 towhich all DNA obtained in Example 1 were introduced. (2) E. coli DH5α/pDXS-1 or E. coli DHS α/pEG400, as obtained in (1) above, wasinoculated into a test tube containing 10 ml of a M9 medium, and thencultured with shaking for 72 hours at 30° C.

After the culture was completed, the amount of CoQ8 produced by thetransformants was calculated in the same manner as in (1) above.

Table 2 shows the results. TABLE 2 CoQ8 Production by transformant ofEscherichia coli Cell Amount Amount of CoQ8 Intracellular Transformant(OD660) Produced (mg/L) Content*¹ E. coli 3.1 0.49 1.6 DH5 α/EG400 E.coli 2.5 1.02 4.1 DH5 α/pDXS-1*¹Intracellular content is shown with a value obtained by dividing a10-fold CoQ8 production (mg/L) by a cell amount (OD660).The amount of CoQ8 produced was significantly higher in DH5 α/pDXS-1than in the control strain DH5 α/pEG400. (3) Production of CoQ8 usingRecombinant Escherichia coli

The plasmid pEGYM1 obtained in Example 1 or pEG400 as a control wasintroduced into E. coli DH5 α and E. coli DH5 α/pEGYM1 and E. coli DH5α/pEG400 that show resistance to spectinomycin at a concentration of 100μg/ml were obtained.

These transformants were inoculated into a test tube containing 10 ml ofLB medium supplemented with 1% glucose, 100 mg/l of vitamin B₁, 100 mg/lof vitamin B₆, 50 mg/l of p-hydroxybenzoic acid. Then the transformantswere cultured with shaking for 72 hours at 30° C.

After the culture was completed, the amount of CoQ8 produced by thetransformants was calculated in the same manner as in (1) above.

Table 3 shows the results. TABLE 3 CoQ8 Production by transformants ofEscherichia coli Cell Amount Amount of CoQ8 Intracellular Transformant(OD660) Produced (mg/L) Content*¹ E. coli 14.44 0.83 0.57 DH5 α/pEG400E. coli 13.12 0.94 0.71 DH5 α/pEGYM1*¹Intracellular content is shown with a value obtained by dividing a10-fold CoQ8 production (mg/L) by a cell amount (OD660).The amount of CoQ8 produced was significantly higher in DH5 α/pEGYM1than in the control strain DH5 α/pEG400.

EXAMPLE 3

Production of Menaquinone-8 (MK-8) by Recombinant Escherichia coli

(1) The E. coli DH5 α/pADO-1 or E. coli DH5 α/pEG400, obtained inExample 2 (1), was inoculated into a test tube containing 10 ml of TBmedium supplemented with 100 μg/ml of spectinomycin, and then culturedwith shaking for 72 hours at 30° C. The TB medium had been prepared bydissolving 12 g of bactotrypton (Difco), 24 g of yeast extract (Difco),and 5 g of glycerol into 900 ml of water followed by the addition of 100ml of aqueous solution containing 0.17 mol/l KH₂PO₄ and 0.72 mol/lK₂HPO₄.

After the culture was completed, MK-8 was quantified in the samequantifying method for CoQ8 as in Example 2 (1), then the amount of MK-8produced by the transformants was calculated.

Table 4 shows the results. TABLE 4 MK-8 Production by transformants ofEscherichia coli Cell Amount Amount of MK-8 Intracellular Transformant(OD660) Produced (mg/L) Content*¹ E. coli 23.2 1.1 0.46 DH5 α/pEG400 E.coli 23.5 1.8 0.75 DH5 α/ADO-1*¹Intracellular content is shown with a value obtained by dividing a10-fold CoQ8 production amount (mg/L) by a cell amount (OD660).The amount of MK-8 produced was significantly higher in DH5 α/pADO-1than in the control DH5 α/pEG400.(2) E. coli DH5 α/pDXS-1 or E. coli DH5 α/pEG400, obtained in Example 2(1), was cultured in the same manner in (1) above, then the amount ofMK-8 produced by the transformants was calculated.

Table 5 shows the results. TABLE 5 Production of MK-8 by transformantsof Escherichia coli Cell Amount Amount of MK-8 IntracellularTransformant (OD660) Produced (mg/L) Control*¹ E. coli 42.8 2.41 0.56DHS α/pEG400 E. coli 44.0 2.96 0.67 DH5 α/pDXS-1*¹Intracellular content is shown with a value obtained by dividing a10-fold CoQ8 production (mg/L) by a cell amount (OD660).The amount of MK-8 produced was significantly higher in DH5 α/pDXS-1than in the control strain DH5 α/pEG400.

EXAMPLE 4 Production of CoQ8 by Recombinant Erwinia carotovora

A plasmid pDXS-1 obtained in Example 1 or pEG400 as a control, wasintroduced into Erwinia carotovora IFO-3380, thereby obtainingtransformants IFO-3380/pDXS-1 and IFO-3380/pEG400, both of which wereresistant to spectinomycin at a concentration of 100 μg/ml.

These transformants were inoculated into a test tube containing 10 ml ofLB medium supplemented with 100 μg/ml of spectinomycin, and thencultured with shaking for 72 hours at 30° C.

After the culture was completed, the amount of CoQ8 produced by thetransformants was calculated in the same manner as in Example 2 (1).

Table 6 shows the results. TABLE 6 CoQ8 Production by transformants ofErwinia carotovora Cell Amount Amount of CoQ8 Intracellular Transformant(OD660) Produced (mg/L) Content*¹ IFO-3380/Peg400 1.68 0.26 1.5Ifo-3380/Pdxs-1 2.48 0.45 1.8*¹Intracellular content is shown with a value obtained by dividing a10-fold CoQ8 production (mg/L) by a cell amount (OD660).The amount of CoQ8 produced was significantly higher in IFO-3380/pDXS-1than in the control strain IFO-3380/pEG400.

EXAMPLE 5 Production of Ubiquinone and Carotenoids by RecombinantErwinia uredovora

The plasmids pUCYM-1, pQEDXS-1, pQEYM-1, obtained in Example 1, or pUC19and pQE30 as controls were introduced into Erwinia uredovora DSM-30080by electroporation, and then the transformants, E. uredovoraDSM-30080/pUCYM-1, E. uredovora DSM-30080/pQEDXS-1, E. uredovoraDSM-30080/pQEYM-1, E. uredovora DSM-30080/pUC19 and E. uredovoraDSM-30080/pQE30, which showed resistant to ampicillin at a concentrationof 100 μg/ml were obtained.

These transformants were inoculated into a test tube containing 10 ml ofLB medium supplemented with 100 μg/ml of ampicillin, 1% glucose, vitaminB₁ and vitamin B₆, 100 mg/l each, and 50 mg/l of p-hydroxybenzoic acid.Then the transformants were cultured by shaking for 72 hours at 30° C.

After the culture was completed, the amount of CoQ8 produced by thetransformants was calculated in the same manner as in Example 2 (1).

The produced amount of carotenoid pigments was calculated by detectingthe absorbance at 450 nm for the 2-butanol layer using aspectrophotometer in the same manner as in Example 2 (1).

Table 7 shows the results. TABLE 7 Production of CoQ8 and Carotenoids bytransformants of E. uredovora CoQ8 Carotenoids IntracellularIntracellular Cell content ratio Production content ratio amountProduction Relative Relative Relative Transformants OD660 mg/L valuevalue value DSM-30080/pUC19 2.00 1.15 1.0 1.0 1.0 DSM-30080/pUCYM-1 1.881.39 1.3 1.5 1.6 DSM-30080/pQE30 2.52 1.29 1.0 1.0 1.0 DSM-30080/pQEYM-11.92 1.36 1.4 1.7 2.2 DSM-30080/pQEDXS-1 2.12 3.21 3.0 5.6 6.7

Both CoQ8 production and carotenoid pigment production weresignificantly higher in DSM-30080/pUCYM-1 than in the control strainDSM-30080/pUC19.

Similarly, both CoQ8 production and carotenoid pigment production weresignificantly higher in DSM-30080/pQEYM-1 and DSM-30080/pQEDXS-1 than inthe control strain DSM-30080/pQE30.

EXAMPLE 6 Cloning of the DNA Encoding Proteins Involved in theBiosynthesis of Isoprenoid Compounds From a Photosynthetic BacteriumRhodobacter sphaeroides

(1) Cloning of DXS Gene From R. sphaeroides

The Genbank database was searched for DXS homologue conserved in otherspecies using the DXS nucleotide sequence found in E. coli. As a result,DXS homologues were found in Haemophilus influenzae (P45205),Rhodobacter capsulatus (P26242), Bacillus subtilis (P54523),Synechocystis sp. PCC6803 (P73067) and Mycobacterium tuberculosis(007184) and the like. Highly conserved amino acid sequences wereselected by comparison of these sequences. A nucleotide sequencecorresponding to such a conserved amino acid sequence was designed inconsideration of the codon usuage in R. sphaeroides. A DNA fragmenthaving a nucleotide sequence of SEQ ID NO:32 and of SEQ ID NO:33, and aDNA fragment having a nucleotide sequence of SEQ ID NO:34 weresynthesized by DNA synthesizer.

PCR was carried out with DNA Thermal Cycler (Perkin Elmer Instruments,Inc. Japan) using chromosomal DNA of R. sphaeroides KY4113 (FERM-P4675)as a template, the primers above, and an Expand™ High-Fidelity PCRSystem (Boehringer Manheim K.K.).

PCR was carried out by 30 cycles, one cycle consisting of reaction at94° C. for 40 seconds, reaction at 60° C. for 40 seconds, reaction at72° C. for 1 minute, followed by reaction at 72° C. for 1 minute,thereby obtaining the DNA fragment of interest. The DNA fragments wereDIG-labeled using DIG DNA Labeling Kit (Boehringer Manheim K.K.).

To obtain the full-length DXS gene of R. sphaeroides, a genomic DNAlibrary of a strain KY4113 was constructed. The strain KY4113 wascultured overnight in LB medium, extracting the chromosomal DNA. Thechromosomal DNA was partially digested with a restriction enzyme Sau3AI,and then 4 to 6 kb DNA fragments were purified by sucrosedensity-gradient centrifugation. The DNA fragments were ligated withBamH I-digested vector pUC19 using a Ligation Pack (Nippon Gene), and E.coli DH5 α was transformed using the ligated DNA. The transformants werespread on LB agar medium containing 100 μg/ml of ampicillin, thusobtaining about 10,000 colonies. As a result of screening by colonyhybridization using the DIG-labeled DNA fragment as a probe, which hadbeen obtained by the above method, two types of DNA fragments weredetected.

As a result of sequencing, ORF sharing high degrees of sequence homologywith known DXS gene of other species was found from each DNA fragment.An amino acid sequence of SEQ ID NO:26 was named DXS1 and that of SEQ IDNO:27 was named DXS2.

(2) Confirmation of Complementarity Using E. coli DXS Gene-DeletedMutant

Selection of E. coli DXS Gene-Deleted Strain

E. coli W3110 (ATCC14948) was inoculated into LB liquid medium, and thencultured to its logarithmic growth phase. After culturing, cells werecollected from the culture by centrifugation.

The cells were washed with 0.05 mol/l Tris-maleate buffer (pH 6.0) andsuspended in the same buffer to a cell density of 10⁹ cells/ml.

NTG was added to the suspension to a final concentration of 600 mg/l,then the mixture was maintained for 20 minutes at room temperature toinduce mutation.

The resultant NTG-treated cells were spread on a M9 minimum agar medium(Molecular Cloning, Second Edition) plate containing 0.1%1-deoxyxylulose, then cultured. 1-Deoxyxylulose had been chemicallysynthesized according to the method described in J. C. S. Perkin TransI, 2131-2137 (1982).

Colonies grew on M9 minimum agar medium containing 0.1% 1-deoxyxylulosewere replicated on M9 minimal agar medium and on M9 minimal agar mediumcontaining 0.1% 1-deoxyxylulose. The mutant of interest, a strainrequiring 1-deoxyxylulose to grow, was selected. That is, a straincapable of growing on minimal agar medium containing 1-deoxyxylulose butnot on the same medium lacking 1-deoxyxylulose was selected.

The thus selected and obtained mutant was named ME1.

When pDXS-1 was introduced into the strain ME1, deficiency in1-deoxyxylulose of the strain ME1 was complemented. Therefore the strainME1 was confirmed to be a strain from which DXS gene was deleted.

(3) Complementation Studies on DXS 1 and DXS2

DNA fragment encoding DXS1 of SEQ ID NO:27 or a DNA fragment encodingDXS2 of SEQ ID NO:29, respectively, both derived from the strain KY4113,was ligated to downstream of the lac promoter of a vector pUC19respectively to construct recombinant plasmids.

When the constructed plasmids were introduced into the strain ME1, bothDXS1 and DXS2 each complemented the 1-deoxyxylulose-deficiency in thestrain ME 1.

Therefore, R. sphaeroides was shown to have two genes, DXS1 and DXS2,having activity to catalyze the reaction to produce 1-deoxy-D-xylulose5-phosphate from pyruvic acid and glyceraldehyde 3-phosphate.

(4) Cloning of Gene Complementing Methylerythritol-Requiring NatureDerived From R. sphaeroides

The E. coli Methylerythritol-requiring mutant ME7 obtained in Example1(2)

was inoculated into LB liquid medium containing 0.1% methylerythritol,cultured to its logarithmic growth phase, then centrifuged to collectcells.

The cells were washed twice with 1 mol/HEPES aqueous solution containing10% glycerol so as to remove the medium components as far as possible.

Plasmids were extracted from the genomic library of R. sphaeroidesKY4113 constructed in Example 6 (1). Then the plasmids were introducedinto the washed cells by electroporation according to standardtechniques.

Next, the cells were spread on LB agar medium containing 100 μg/l ofampicillin, then cultured overnight at 37° C.

After picking up the colonies grown on the medium, the colonies wereinoculated into LB liquid medium to culture, then plasmids wereextracted from the cells cultured.

When the plasmids extracted were introduced again into the strain ME 7,the transformants could grow in a medium lacking methylerythritol.Therefore it was confirmed that the plasmid contained a DNA fragmentcomplementing methylerythritol-requiring nature derived from R.sphaeroides.

As a result of sequencing of the nucleotide sequence of the DNAfragment, the DNA sequence of SEQ ID NO:31 encoding an amino acidsequence that shares high homology with E. coli yaeM was found.

EXAMPLE 7 Production of Ubiquinone-10 (CoQ10) By RecombinantPhotosynthetic Bacteria

A glnB promoter derived from the strain KY4113 was ligated upstream ofthe DNA fragment DXS1 of SEQ ID NO:27 and DXS2 of SEQ ID NO:29, bothobtained in Example 6. Then the product was inserted into a broad hostrange vector pEG400, thus constructing plasmids. These plasmids werenamed pRSDX-1 and pRSDX-2, respectively. In addition, yaeM and DXS1 werejoined in tandem, then the product was ligated downstream of ginBpromoter, thereby constructing a plasmid. The plasmid was namedpRSYMDX1. These plasmids were introduced into R. sphaeroides KY4113,respectively, by electroporation (Bio-Rad Laboratories).

Then the cells were spread on LB agar medium containing spectinomycin ata concentration of 100 μg/ml, then cultured for 3 days at 30° C.

Next, colonies that grew on the medium were inoculated into LB mediumcontaining spectinomycin at a concentration of 100 μg/ml, culturedovemight. Then, the cultured cells were collected by centrifugation.

It was confirned that the cells of each strain contained the introducedplasmid by extracting the plasmids from the cells (Qiagen, Inc). Thusobtained transformants were named KY4113/pRSDX-1, KY4113/pRSDX-2,KY4113/pRSYMDX1 and KY4113/pEG400.

A platinum loop of each transformant was inoculated into a test tubecontaining 5 ml of seed medium (2% glucose, 1% peptone, 1% yeastextract, 0.5% NaCl, pH 7.2 adjusted with NaOH) and then cultured for 24hours at 30° C.

0.5 ml of the resultant culture was inoculated into a test tubecontaining 5 ml of ubiquinone-10 production medium, then cultured byshaking for 5 days at 30° C. The ubiquinone-10 production mediumconsisted of 4% blackstrap molasses, 2.7% glucose, 4% corn steep liquor,0.8% ammonium sulfate, 0.05% potassium primary phosphate, 0.05%potassium secondary phosphate, 0.025% magnesium sulfate heptahydrate, 3mg/l of ferrous sulfate heptahydrate, 8 mg/l of thiamine, 8 mg/l ofnicotinic acid, and 1 ml/l of trace element, had previously beenadjusted to pH 9, supplemented with 1% calcium carbonate, thenautoclaved.

Then the amount of CoQ10 produced by the transformants was calculated inthe same manner as in quantification of CoQ8 in Example 2 (1). Table 8shows the results. TABLE 8 Amount of CoQ10 Cell amount [OD660]Accumulated [mg/l] KY4113/pEG400 23.7 65.2 KY4113/pRSDX-1 23 81KY4113/pRSDX-2 24.4 81.9 KY4113/pRSYMDX1 25.8 117.9The amount of CoQIO produced was significantly higher in KY4113/pRSDX-1,KY4113/pRSDX-2 and KY4113/pRSYMDX1 than in the control strainKY4113/pEG400.

EXAMPLE 8 Determination of the Activity of the Enzyme Encoded By yaeMGene

(1) Overexpression of yaeM Gene

A recombinant plasmid that can express yaeM gene sufficiently wasconstructed using PCR [Science, 230, 1350 (1985)], as follows.

A sense primer having a nucleotide sequence of SEQ ID NO:24 and anantisense primer having a nucleotide sequence of SEQ ID NO:25 weresynthesized using a DNA synthesizer.

A restriction enzyme BamH I site was added to each of 5′-ends of thesense and antisense primers.

yaeM gene was amplified by PCR using chromosomal DNA of E. coli as atemplate, these primers, Taq DNA polymerase (Boehringer), and DNAThermal cycler (Perkin Elmer Japan).

PCR was carried out by 30 cycles, one cycle consisting of reaction at94° C. for 30 seconds, reaction at 55° C. for 30 seconds, and reactionat 72° C. for 2 minutes followed by reaction at 72° C. for 7 rninutes.

The amplified DNA fragments and pUC118 (TAKARA SHUZO Co., Ltd.) weredigested with a restriction enzyme BamH I, then each DNA fragment waspurified by agarose gel electrophoresis.

Both purified fragments were mixed together, then treated with ethanol,allowing DNA to precipitate. The resultant DNA precipitate was dissolvedin 5 μl of distilled water for ligation reaction to occur, therebyobtaining recombinant DNA.

The recombinant DNA was confirmed to be yaeM gene by determining its DNAsequence.

Plasmids were extracted from the microorganism having the recombinantDNA, digested with a restriction enzyme BamH I, and subjected to agarosegel electrophoresis, thereby obtaining DNA fragments containing BamHI-treated yaeM gene.

pQE30 (Qiagen, Inc) was digested with a restriction enzyme BamH I, thensubjected to agarose gel electrophoresis, thereby obtaining BamHI-treated pQE30 fragments.

The resultant DNA fragments containing BamH I-treated yaeM gene weremixed with BamH I-digested pQE30 fragments, and treated with ethanol forDNA to precipitate. The DNA precipitate was dissolved in 5 μl ofdistilled water for ligation reaction to occur, thereby obtainingrecombinant DNA.

E. coli JM109 was transformed using the recombinant DNA by standardtechniques. Then the transformants were spread on LB agar mediumcontaining 100 μg/ml of ampicillin, then cultured overnight at 37° C.

Plasmids were isolated from the E. coli in the same manner as describedabove.

Similarly, the isolated plasmid was cleaved with various restrictionenzymes to examine the structure, then the nucleotide sequence wasdetermined, thereby confirming the plasmids contained the DNA fragmentsof interest. The plasmid was named pQEDXR.

(2) Determination of Activity of yaeM Gene Product

Purification of yaeM Gene Product

The pQEDXR constructed in (1) was introduced into E. coli M15 (Qiagen,Inc) having pREP4 by standard techniques, and a strain M15/pREP4+pQEDXRresistant to 200 μg/ml of ampicillin and 25 μg/ml of kanamycin wasobtained.

The strain M15/pREP4+pQEDXR was cultured at 37° C. in 100 ml of LBliquid medium containing 200 μg/ml of ampicillin and 25 μg/ml ofkanamycin. When the turbidity at 660 nm reached 0.8, isopropylthiogalactoside was added to a final concentration of 0.2 mol/l.Subsequently, the strain was cultured for 5 hours at 37° C., then thesupernatant of the culture was removed by centrifugation (3000 rpm, 10minutes). The cells were suspended in 6 ml of 100 mol/lTris-hydrochloric acid buffer (pH 8.0), then disrupted using aultrasonicator (SONIFIER, BRANSON) while cooling with ice. The obtainedcell-disrupted solution was centrifuged at 10,000 rpm for 20 minutes at4° C., thereby collecting the supernatant. The supernatant centrifugedfrom the cellular extract was introduced into a Ni-NTA resin column(Qiagen, Inc), then washed with 20 ml of a washing buffer (100 mol/lTris-hydrochloric acid (pH 8.0), 50 mol/l imidazole, 0.5% Tween 20).Then 10 ml of an elution buffer (100 mol/l Tris-hydrochloric acid (pH8.0), 200 mol/l imidazole) was introduced into the column, thusfractionating the eluate into 1 ml each.

Protein amounts for each fraction were measured using a kit forquantifying protein amount (Bio-Rad Laboratories), thus obtaining afraction containing proteins as a purified protein fraction.

Preparation of a Substrate, 1-Deoxy-D-xylulose 5-Phosphate

A reaction substrate, 1-deoxy-D-xylulose 5-phosphate was prepared asdescribed below. 1-Deoxy-D-xylulose 5-phosphate was detected bymeasuring the absorbance at 195 nm using HPLC [Column: Senshu pakNH2-1251-N (4.6×250 mm, Senshu), mobile phase:100 mol/l KH₂PO₄ (pH3.5)].

The plasmid pQDXS-1 that allows overexpression of E. coli dxs gene wasintroduced into E. coli M15/pREP4 in the same manner as described above,obtaining a strain M15/pREP4+pQDXS-1.

This strain was cultured in the same way as in Example 8 (2)

, then dxs protein was purified using Ni-NTA resin column.

The purified dxs protein was added to a 20 ml of reaction solution [100mol/l Tris-hydrochloric acid (pH 7.5), 10 mol/l sodium pyruvate, 30 moliDL-glyceraldehyde-3-phosphate, 1.5 mol/l thiamine pyruvate, 10 mol/lMgCI₂, 1 mol/l DL-dithiothreitol] then maintained at 37° C.

After reacting for 12 hours, the reaction solution was diluted withwater to 300 ml, introduced into an activated carbon column (2.2×8 cm)followed by a Dowex 1-X8 (C1-type, 3.5×25 cm), then eluted with 1%saline solution. After the eluted fraction was concentrated, thefraction was introduced into Sephadex G-10 (1.8×100 cm), then elutedwith water. Finally fractions containing 1-deoxy-D-xylulose 5-phosphatewere freeze-dried, thereby obtaining about 50 mg of white powder.

This powder was confirmed to be 1-deoxy-D-xylulose 5-phosphate by NMRanalysis (A-500, JEOL Ltd.).

Determiination of Enzymatic Activity of yaeM Gene Product

0.3 mol/l of 1-deoxy-D-xylulose 5-phosphate (final concentration)synthesized as described above was added to 1 ml of a reaction solutioncontaining 100 mol/l Tris-hydrochloric acid (pH 7.5), 1 mol/l MmCl₂, 0.3mol/l NADPH and yaeM gene product obtained in Example 8 (2)

, and then incubated at 37° C. The increase and decrease in NADPH duringincubation was traced by reading the absorbance at 340 nm using aspectrophotometer (UV-160, SHIMADZU CORP.), suggesting that NADPHdecreased with time.

To confirm the structure of the reaction product, the reaction wascarried out similarly, but on a larger scale, thus isolating theproduct. 200 ml of a reaction solution with a composition, the same asthat described above except that the concentration of 1-deoxy-D-xylulose5-phosphate was 0.15 mol/l, was incubated for 30 minutes at 37° C. Thenthe whole amount of the reaction solution was added to an activatedcarbon column, diluted with water to 1 L, then added to a Dowex 1-X8(C1-type, 3.5×20 cm) column.

The solution was eluted with 400 ml of 1% saline solution, added to aSephadex G-10 (1.8×100 cm), then eluted with water. The eluted fractionwas freeze-dried, thereby isolating the reaction product.

The molecular formula of the reaction product isolated from HR-FABMSanalysis was assumed to be C₅H₁₂O₇ P [m/z 215.0276 (M-H), Δ-4.5 mmu].NMR analysis for ¹H and ¹³C resulted in the following chemical shifts.

¹H NMR (D₂O, 500 MHz): δ 4.03 (ddd, J=11.5, 6.5, 2.5 Hz, 1H), 3.84 (ddd,J=11.5, 8.0, 6.5 Hz, 1H), 3.78 (dd, J=80, 2.5 Hz, 1H), 3.60 (d, J=12.0Hz, 1H), 3.50 (d, J=12.0 Hz, 1H), 1.15 (s, 3H); ¹³C NMR (D₂O, 125 MHz):δ 75.1 (C-2), 74.8 (C-3), 67.4 (C-1), 65.9 (C-4), 19.4 (2-Me)

The chemical shifts resulted from NMR analysis for ¹H and ¹³C ofcompounds obtained by treating the reaction products with alkalinephosphatase (TAKARA SHUZO CO., LTD.) were completely identical with thatresulted from NMR analysis of 2-C-methyl-D-erythritol synthesized in themethod described in Tetrahedron Letter, 38, 6184 (1997).

Further the angle of rotation of the former compound was [α]_(D) ²¹=+6.0(c=0.050, H₂O), identical with the angle of rotation [α]_(D) ²⁵=+7.0(c=0.13, H₂O) of 2-C-methyl-D-erythritol, reported in TetrahedronLetter, 38, 6184 (1997).

These results reveal that the reaction product of yaeM gene product was2-C-methyl-D-erythritol 4-phosphate. That is, yaeM gene product wasfound to have activity to yield 2-C-mehyl-D-erythritol 4-phosphate fromldeoxy-D-xylulose 5-phosphate with consumption of NADPH. Based on thiscatalytic activity, this enzyme was named 1-deoxy-D-xylulose 5-phosphatereductoisomerase.

Characteristics of 1-deoxy-D-xylulose 5-Phosphate Reductoisomerase

The enzymological characteristics of 1-deoxy-D-xylulose 5-phosphatereductoisomerase were examined using 1 ml of the reaction system asdescribed in Example 8 (2)

. Here, 1 unit is defined as the activity to oxidize 1 mmol of NADPH pera minute.

The activity decreased below 1/100 when NADPH was replaced with NADH.

No reaction occurred when 1-deoxy-D-xylulose was used instead of1-deoxy-D-xylulose 5-phosphate.

SDS-PAGE analysis showed that this enzyme was consisted of 42 kDapolypeptide.

Table 9 shows effect on the reaction system by the addition of metals.TABLE 9 Effect of various metal ions on the activity of1-deoxy-D-xylulose 5-phosphate reductoisomerase Specific ActivityAdditives (units/mg protein) none 0.3 EDTA N.D. MnCl₂ 11.8  CoCl₂ 6.0MgCl₂ 4.0 CaCl₂ 0.2 NiSO₄ 0.2 ZnSO₄ 0.3 CuSO₄ N.D. FeSO₄ N.D.

These metal ions and EDTA were added such that the concentration of eachwas 1 mol/l. N.D. indicates that no activity was detected.

Km for 1-deoxy-D-xylulose 5-phosphate and NADP in the presence of MnCl₂were 249 μmol/l and 7.4 μmol/l, respectively.

FIG. 1 shows the effect of reaction temperature and FIG. 2 shows theeffect of reaction pH.

EXAMPLE 9 Construction and Characteristics of yaeM-Deleted Mutant

(1) Construction of yaeM-Disrupted Mutant

To test whether 1-deoxy-D-xylulose 5-phosphate reductoisomerase isessential for cell growth or not, a 1-deoxy-D-xylulose 5-phosphatereductoisomerase-deleted mutant was constructed as described below.

A kanamycin-resistant gene cassette for insertion into yaeM gene wasproduced as described below.

The plasmid pMEW41 obtained in Example 1 (2)

was digested with a restriction enzyme Bal I, and was subjected toagarose gel electrophoresis, thereby obtaining a Bal I-treated DNAfragment.

Tn5 was digested with restriction enzymes Hind III and Sam I, then theboth ends were blunt-ended using a DNA blunting kit (TAKARA SHUZO CO.,LTD.).

The resultant blunt-ended DNA fragments were mixed with previouslyobtained Bal I-treated pMEW41 DNA fragments, and then the mixture wastreated with ethanol. Next the obtained DNA precipitate was dissolvedinto 5 μl of distilled water for ligation reaction to occur, therebyobtaining recombinant DNA.

E. coli JM109 (purchased from TAKARA SHUZO CO., LTD.) was transformedusing this recombinant DNA according to standard techniques. Next thetransformant was spread on LB agar medium containing 100 μg/ml ofampicillin and 15 μg/ml of kanamycin, then cultured overnight at 37° C.

Several ampicillin-resistant transformant colonies grown on the mediumwere shake-cultured for 16 hours at 37° C. in 10 ml of LB liquid mediumcontaining 100 μg/ml of ampicillin and 15 μg/ml of kanamycin.

The resulting culture was centrifuged to collect cells.

Plasmids were isolated from the cells according to the standardtechniques.

The plasmids isolated as described above were cleaved with variousrestriction enzymes to test their structure. As a result, the plasmidwas confirmed to contain the DNA fragment of interest and was namedpMEW41Km.

yaeM gene on a chromosomal DNA of E. coli was disrupted by homologousrecombination using pMEW41Km. FIG. 3 shows the schematic diagram forthis recombination.

pMEW41Km was digested with restriction enzymes Hind III and Sac I,subjected to agarose gel electrophoresis, thus purifying linearfragments. E. coli FS1576 was transformed using the fragments accordingto standard techniques. The strain FS1576 is available as the strainME9019 from National Institute of Genetics. The transformants werespread on LB agar medium containing 15 μg/ml of kanamycin and 1 g/l of2-C-methyl-D-erythritol, then cultured overnight at 37° C.

Several kanamycin-resistant colonies that grew on the medium wereshake-cultured for 16 hours at 37° C. in 10 ml of LB liquid mediumcontaining 15 μg/ml of kanamycin and 1 g/l of 2-C-methyl-D-erythritol.

The resulting culture was centrifuged to collect cells.

Chromosomal DNA was isolated from the cells by the standard techniques.

The chromosomal DNA was digested with a restriction enzyme Sma I or PstI. Chromosomal DNA of the strain FS1576 was digested with a restrictionenzyme in the same way. These DNAs digested with restriction enzymeswere subjected to agarose gel electrophoresis by the standardtechniques, and then to Southern hybridization analysis using thekanamycin-resistant gene and yaeM gene as probes. Therefore, it wasconfirmed that the chromosomal DNA of the kanamycin-resistant colonieshad a structure as shown in FIG. 3, that is, yaeM gene was disrupted bythe kanamycin-resistant gene.

(2) Characteristics of yaeM-Disrupted Mutant

The yaeM-disrupted strain produced as described above and its parentstrain FS1576 were spread on LB agar medium and the same mediumcontaining 1 g/l of 2-C-methyl-D-erythritol, then cultured at 37° C.Table 10 shows the cell growth after 2 days of culture. TABLE 10 Effectof deletion of yaeM gene on the E. coli growth Cell growth on eachmedium Strain LB LB + ME*² FS1576 + + yaeM-deleted strain − +*¹Cell growth (+ indicates good growth; − indicates no growth)*²ME indicates the addition of 1 g/l of 2-C-methyl-D-erythritol.

No yaeM-deleted mutants grew on a medium lacking2-C-methyl-D-erythritol. Therefore, This gene was shown to be essentialfor the cell growth in the absence of 2-C-methyl-D-erythritol.

EXAMPLE 10 Effect of 1-Deoxy-D-xylulose 5-Phosphate ReductoisomeraseInhibitor for Cell Growth

The following experiments were conducted based on the assumption thatfosmidomycin could inhibit 1-deoxy-D-xylulose 5-phosphatereductoisomerase because 2-C-methyl-D-erythritol 4-phosphate, a productfrom 1-deoxy-D-xylulose 5-phosphate reductoisomerase reaction, orreaction intermediates expected to be produced in this enzyme reactionis structurally analogous to fosmidomycin.

In the presence of fosmidomycin, the activity 1-deoxy-D-xylulose5-phosphate reductoisomerase was measured by the method as described inExample 8 in order to examine the effect on the enzymatic activity.

Fosmidomycin had been synthesized according to the method described inChem. Pharm. Bull, 30, 111-118 (1982).

Total volume of reaction solution was reduced to 0.2 ml from the volumeof reaction solution described in Example 8 (2), but each concentrationwas kept at the same level as the system of Example 8

. Fosmidomycin at various concentration was added to the reactionsolution, then the reaction was carried out at 37° C. The increase anddecrease in NADPH were measured using Bench mark micro plate reader(Bio-Rad Laboratories).

As shown in FIG. 4, fosmidomycin was shown to inhibit 1-deoxy-D-xylulose5-phosphate reductoisomerase.

E. coli W3110 was spread on LB agar medium, the same medium containing3.13 mg/l of fosmidomycin, and the same medium containing 3.13 mg/l offosmidomycin and 0.25 g/l of 2-C-methyl-D-erythritol, then cultured at37° C.

Two days after culturing, the microorganism could grow on the two typesof media, that is, the LB agar medium and the same medium containingfosmidomycin and 0.25 g/l of 2-C-methyl-D-erythritol, but nomicroorganism grew on the LB agar medium supplemented only withfosmidomycin.

These results clearly shows that fosmidomycin inhibited the cell growthby inhibiting 1-deoxy-D-xylulose 5-phosphate reductoisomerase.Accordingly, a substance inhibiting yaeM gene product(1-deoxy-D-xylulose 5-phosphate reductoisomerase) activity can be aneffective antibiotic agent or herbicide.

All publications, patents and patent applications cited herein areincorporated herein by reference in their entirety.

INDUSTRIAL APPLICABILITY

The present invention can provide a process for producing isoprenoidcompounds comprising integrating DNA into a vector wherein the DNAcontains one or more DNA involved in biosynthesis of isoprenoidcompounds useful in pharmaceuticals for cardiac diseases, osteoporosis,homeostasis, prevention of cancer, and immunopotentiation, health foodand anti-fouling paint products against barnacles, introducing theresultant recombinant DNA into a host cell derived from prokaryote,culturing the obtained transformants in a medium, allowing thetransformant to produce and accumulate isoprenoid compounds in theculture, and recovering the isoprenoid compounds from the culture, aprocess for producing a protein having activity to improve efficiency inthe biosynthesis of isoprenoid compounds comprising integrating DNAcontaining one or more DNA encoding the protein into a vector,introducing the resultant recombinant DNA into a host cell, culturingthe obtained transformant in a medium, allowing the transformant toproduce and accumulate said protein in the culture, and recovering saidprotein from the culture; the protein; and novel enzymatic proteinhaving activity to catalyze a reaction to produce2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate;and a method for screening a compound with antibiotic and/or weedingactivity comprising screening a substance inhibiting the enzyme.

SEQUENCE LISTING FREE TEXT

-   SEQ ID NO:12: synthetic DNA-   SEQ ID NO:13: synthetic DNA-   SEQ ID NO:14: synthetic DNA-   SEQ ID NO:15: synthetic DNA-   SEQ ID NO:16: synthetic DNA-   SEQ ID NO:17: synthetic DNA-   SEQ ID NO:18: synthetic DNA-   SEQ ID NO:19: synthetic DNA-   SEQ ID NO:20: synthetic DNA-   SEQ ID NO:21: synthetic DNA-   SEQ ID NO:22: synthetic DNA-   SEQ ID NO:23: synthetic DNA-   SEQ ID NO:24: synthetic DNA-   SEQ ID NO:25: synthetic DNA-   SEQ ID NO:32: synthetic DNA-   SEQ ID NO:33: synthetic DNA-   SEQ ID NO:34: synthetic DNA

1-22. (canceled)
 23. A process for producing an isoprenoid compound comprising the steps of: introducing a vector containing DNA encoding a protein comprising an amino acid sequence of SEQ ID NO:26 into a prokaryotic host cell to produce a transformant; culturing the transformant in a medium; allowing the transformant to produce and accumulate the isoprenoid compound; and recovering the isoprenoid compound.
 24. A process for producing an isoprenoid compound comprising the steps of: culturing a prokaryotic transformant harboring a vector containing DNA encoding a protein comprising an amino acid sequence of SEQ ID NO:26 in a medium; allowing the transformant to produce and accumulate the isoprenoid compound; and recovering the isoprenoid compound.
 25. The process according to claim 23 or 24, wherein the DNA comprises a nucleotide sequence of SEQ ID NO:27.
 26. The process according to claim 23 or 24, wherein the isoprenoid compound is selected from the group consisting of ubiquinone, vitamin K₂ and carotenoid.
 27. The process according to claim 25, wherein the isoprenoid compound is selected from the group consisting of ubiquinone, vitamin K₂ and carotenoid.
 28. A process for producing an isoprenoid compound comprising the steps of: introducing a vector containing DNA which hybridizes with a nucleotide sequence consisting of SEQ ID NO:27 in the presence of 0.7 to 1.0 mol/l NaCl at 65° C. followed by washing in a 0.1 to 2-fold SSC solution at 65° C. and encodes a protein having activity to catalyze a reaction to produce 2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate into a prokaryotic host cell to produce a transformant; culturing the transformant in a medium; allowing the transformant to produce and accumulate the isoprenoid compound; and recovering the isoprenoid compound.
 29. A process for producing an isoprenoid compound comprising the steps of: culturing a prokaryotic transformant harboring a vector containing DNA that hybridizes with a nucleotide sequence consisting of SEQ ID NO:27 in the presence of 0.7 to 1.0 mol/l NaCl at 65° C. followed by washing in a 0.1 to 2-fold SSC solution at 65° C. and encodes a protein having activity to catalyze a reaction to produce 2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate in a medium; allowing the transformant to produce and accumulate the isoprenoid compound; and recovering the isoprenoid compound.
 30. The process according to claim 28 or 29, wherein the isoprenoid compound is selected from the group consisting of ubiquinone, vitamin K₂ and carotenoid. 